Use of acetyl-coa carboxylase for identifying compounds that have an insecticidal effect

ABSTRACT

The invention relates to nucleic acids which encode insect polypeptides with the biological activity of acetyl-CoA carboxylases, to the polypeptides encoded by them, and to their use for identifying novel, insecticidally active compounds. The invention furthermore relates to methods of finding modulators of these polypeptides, and to the use of these compounds as inhibitors of insect ACCase.

[0001] The invention relates to the use of polypeptides and enzymepreparations with the biological activity of an acetyl-CoA carboxylasefor identifying new, insecticidally active compounds, and to methods offinding modulators of these polypeptides.

[0002] Acetyl-CoA carboxylase (EC 6.4.1.2), hereinbelow referred to asACCase, catalyzes the biotin-dependent carboxylation of acetyl-CoA andis the pacemaker of the de novo fatty acid biosynthesis. ACCase hasthree domains: the biotin-carboxyl carrier (BCC), biotin carboxylase(BCase) and carboxyltransferase (CTase). The ACCase-catalyzed reactioncan be divided into two steps. In a first step, a CO₂ group istransferred from bicarbonate, by the BCase activity, to a biotin whichis bonded covalently to the BCC, with ATP cleavage. In the next step,the carboxyl group activated thus is transferred to acetyl-CoA by CTase,with the formation of malonyl-CoA (Knowles J R, 1989). There are two,physiologically different, forms of ACCase. In the heteromeric form,which is found in bacteria and in the chloroplasts of plants, threedomains are formed by three separate, dissociable proteins. Thehomomeric ACCase consists of a polypeptide chain comprising all threedomains and which is found in the cytosol of plants, animals and fungi(Ke J et al., 2000). In plants and vertebrates, ACCase is regulated by alarge number of mechanisms, for example allosterically by citrate,palmitoyl-CoA, by phosphorylation/dephosphorylation, by protein kinasesand at the level of gene expression (Munday M R & Hemingway C J 1999; KeJ et al. 2000). No information is available on the regulation of theenzymes from insects.

[0003] A large number of genes of ACCases from plants, fungi andvertebrates have already been cloned (for example Abu-Elheiga L et al.1994; Bailey A et al. 1995; Goffeau A et al. 1996; ACCase from theArabidopsis thaliana Genbank AAF18638546) or applied for as patents (forexample Haselkorn R & Gornicki P 1999; Somers D A 1999; Jenkins A R etal. 1992). However, an annotated sequence from insects, that is to sayone which is assigned to ACCase, is as yet unknown.

[0004] Inhibitors of ACCase from plants and fungi are already known asherbicides or fungicides from a large number of biochemical papers onplants and fungi (Vahlensieck H F et al. 1994; Gronwald J W 1994).Another document describes the fungicides Soraphen A and B, which areknown as ACCase inhibitors, for controlling mites, which do not belongto the order of the insects (Sutter M. et al., 1991).

[0005] The effect of inhibitors of human ACCase on insects was studiedin a publication (Popham, H J R et al. (1996): Effect of a hypolipidemicagent on the growth and development of the southwestern corn borer,Diatraea grandiosella. Comp. Biochem. Physiol., C: Pharmacol., Toxicol.Endocrinol. (1996), 115 (3), 247-249); however, this paper predominantlydeals with aspects of physiology.

[0006] It was therefore an object of the present invention to makeavailable ACCase from insects, to test its suitability as target forinsecticides and to provide methods for identifying insecticidal activecompounds.

[0007] In the present invention, crude extracts have been obtained froma variety of instars or adults of the peach aphid Myzus persicae byhomogenizing them in suitable buffers. These crude extracts werepre-purified, and the ACCase activity was determined in a radioactiveenzyme assay.

[0008] It has now been demonstrated for the first time within the scopeof the present invention that compounds exist which inhibit the activityof ACCase from insects, for example from Myzus persicae, in an enzymeassay. The finding that certain compounds inhibit ACCase shows thatACCase is the target of these active compounds and constitutes a targetprotein of insecticidally active compounds.

[0009] It is furthermore demonstrated within the present invention thatin particular cyclic 1,3-dicarbonyl compounds and their enols of theformula (I)

[0010] in which

[0011] Ar represents substituted aryl or hetaryl having at least oneortho-substituent,

[0012] R represents H, or represents acyl radicals, preferably theradicals COR¹ and CO₂R¹,

[0013] in which

[0014] R¹ represents optionally substituted alkyl, phenyl or hetaryl and

[0015] A together with the linked C atoms forms an optionallysubstituted 5- or 6-membered carbo- or heterocycle, suitable heteroatomsbeing, for example, N, O and/or S,

[0016] constitute ACCase inhibitors. Such cyclic 1,3-dicarbonylcompounds are known from the following documents, which are expresslyincorporated into the present application:

[0017] EP-A-355 599, EP-A-377 893, EP-A415 211, EP-A-442 077, EP-A-442073, EP-A-497 127, EP-A-501 129, EP-A-615 950, EP-A-521 334, EP-A-596298, EP-A-613 884, EP-A-613 885, EP-A-706 527, EP-A-643 159,EP-A-741700, EP-A-668 267, EP-A-754 175, EP-A-792 272, EP-A-809 629,EP-A-825 982, EP-A-835 243, EP-A-837 847, EP-A-891 330, EP-A-912 547,EP-A-915 846, EP-A-918 775, EP-A-944 633, EP-A-1 017 674, EP-A-1 028963, EP-A-1 056 717, WO-A-99/48869, WO-A-99/55673, EP-A-528 156,EP-A-647 637, EP-A-792 272, EP-A-799 228, EP-A-944 633, EP-A-1 017 674,EP-A-588 137, EP-A-799 228, EP-A-751 942, EP-A-588 137, EP-A-879 232,EP-A-865 438, WO-A-00/15632, WO-A-00/21946, WO-A-00/24729, EP-A-675 882,EP-A-769 001, EP-A-987 246, EP-A-773 920, EP-A-854 852, EP-A-966 420,EP-A-508 126.

[0018] It is thus shown in the present application that theabovementioned cyclic 1,3-dicarbonyl compounds can be used as inhibitorsof ACCase in insects. The present invention also provides the use of thecompounds of the formula (I) as inhibitors of ACCase in insects.

[0019] It is furthermore shown in the present invention that theinhibition of ACCase results in the death of treated insects. It washitherto unknown that ACCase in insects is a target protein ofinsecticidally active substances. It is thus also demonstrated for thefirst time that ACCase is a vital enzyme for insects and thereforeparticularly suitable for being used as target protein in the search forfurther, possibly improved, insecticidal active compounds.

[0020] The present invention furthermore describes for the first timethe ACCase from Drosophila melanogaster by its nucleic acid sequence,thus making it available. The nucleic acid sequence of Accession NumberAAF59156 has already been available for some time. However, the meaningof the sequence, or the polypeptide encoded by it and its biologicalfunction, were hitherto unknown, as was the coding region of thissequence segment.

[0021] The meaning, function and coding region, and the polypeptideencoded by this nucleic acid, are now made available for the first timewithin the scope of the present invention. Thus, the cDNA of SEQ IDNO:1, which encodes the Drosophila melanogaster ACCase, and thepolypeptide of SEQ ID NO:2 which is encoded by it are disclosed in thepresent application, and their use for identifying insecticidally and,if appropriate, also acaricidally active substances are described. Thenucleic acid sequence of SEQ ID NO:1 and the polypeptide of SEQ ID NO:2encoded by it are likewise the subject matter of the present invention.

[0022] Since the ACCases, in particular also the present ACCase fromMyzus persicae and Drosophila melanogaster and from other insects showconsiderable homology with each other, it is also possible to usehomologous polypeptides which are encoded by the relevant homologousnucleic acids, and other members of the gene family, as moleculartargets of insecticidal active compounds, in particular of the compoundsof the formula (I). The homologous polypeptides are especiallypreferably those with 60%, preferably 80%, especially preferably 90% andespecially preferably 95% identity with the Myzus persicae or Drosophilamelanogaster ACCase over a length of at least 20, preferably at least25, especially preferably at least 30, consecutive amino acids and veryespecially preferably over the full length.

[0023] Thus, insecticidal and/or acaricidal active compounds which maybe found with the aid of the ACCases according to the invention, arealso capable of interacting with ACCases from a large number of otherAcarina or insect species, but the interaction with the differentACCases which are found in the insects or Acarina need not always beequally pronounced. This explains, inter alia, the observed selectivityof the substances which act on this enzyme. Especially preferredACCases, or their organisms of origin, are listed in Table 1 hereinbelowby way of example, but not by limitation: TABLE 1 Preferred organisms oforigin of the ACCases according to the invention 1 Drosophilamelanogaster 2 Heliothis virescens 3 Mycus persicae

[0024] In the present application, it is shown for the first time asexemplified by the ACCase from the peach aphid Myzus persicae thatACCases are target proteins for insecticidal active compounds and can beused for identifying new, improved insecticidal active compounds insuitable methods (assays).

[0025] The Myzus persicae and Drosophila melanogaster ACCase areparticularly suitable in this context for identifying new insecticidaland, if appropriate, also acaracidal active compounds.

[0026] The present invention therefore relates to the use of insectpolypeptides with the biological activity of an ACCase and to nucleicacids encoding them for identifying ACCase modulators in insects and/orAcarina, in particular of those polypeptides which have been isolateddirectly from insects or which are encoded by nucleic acid sequences orfragments thereof which originate from insects and which are obtained byin-vivo or in-vitro methods. The polypeptides are especially preferablythose which have 60%, preferably 80%, especially preferably 90% and veryespecially preferably 95% identity with the Myzus persicae or Drosophilamelanogaster ACCase over a length of at least 20, preferably at least25, especially preferably at least 30, consecutive amino acids and veryespecially preferably over the full length.

[0027] In particular, the present invention relates to the use of ACCasefrom insects of the families Aphididae and Dipterea.

[0028] In particular, the present invention relates to the use of Myzuspersicae ACCase and of Drosophila melanogaster ACCase as shown in SEQ IDNO: 2, and of homologous polypeptides for identifying insect ACCasemodulators.

[0029] In particular, the present invention relates to the use of Myzuspersicae ACCase for identifying insect ACCase modulators.

[0030] Very especially preferably, the polypeptides according to theinvention thus encompass a sequence selected from

[0031] a) the sequence isolated from Mycus persicae,

[0032] b) the sequence as shown in SEQ ID NO: 2,

[0033] c) the sequence encoded by the nucleic acid of Accession NumberAAF59156,

[0034] d) part-sequences of the sequences mentioned under a) to c) whichretain the biological activity of an ACCase,

[0035] e) sequences which have at least 60%, preferably 80%, especiallypreferably 90% and very especially preferably 95% identity with thesequences mentioned under a) to d).

[0036] The degree of the identity of the amino acid sequences ispreferably determined with the aid of the GAP program from the programpackage GCG, Version 10.0 using standard settings (Devereux et al.1984).

[0037] The present invention also relates to the use of insect nucleicacids encoding ACCases, for identifying ACCase modulators in insectsand/or Acarina.

[0038] In particular, the present invention also relates to the use ofthe nucleic acid encoding the Myzus persicae ACCase and of the nucleicacid encoding the Drosophila melanogaster ACCase as shown in SEQ ID NO:1 for identifying ACCase modulators, and nucleic acid sequences whichhave 60%, preferably 80%, especially preferably 90% and especiallypreferably 95% homology therewith.

[0039] The nucleic acids according to the invention are, in particular,single-stranded or double-stranded deoxyribonucleic acids (DNA) orribonucleic acids (RNA).

[0040] Preferred embodiments are fragments of genomic DNA which maycomprise introns, and cDNAs.

[0041] The term “cDNA” as used in the present context refers to asingle- or double-stranded copy of an RNA molecule and, being the copyof a biologically active RNA, is therefore free from introns, i.e. allcoding regions of the gene are present in contiguous form.

[0042] The term “identity” as used in the present context refers to thenumber of sequence positions which are identical in an alignment. It isusually given as a percentage of the alignment length.

[0043] The term “percent (%) identity” as used in the present contextwhen referring to a specific sequence or a specific part of the sequenceis defined as the percentage of nucleotides in the nucleic acid moleculestudied which is identical with the nucleotides of said specificsequence or a specific part of this sequence when the sequences arecompared with each other (“alignment”) and when, if necessary, what areknown as “gaps” are introduced in order to obtain the maximum percentageof identical sequences, with all parameters of the program used set to“default”.

[0044] The term “similarity” as used in the present context, incontrast, assumes the definition of a similarity metric, that is to saya measure for the desired assumed similarity between, for example, avaline and a threonine or a leucine.

[0045] The term “percentage (%) similarity”, as used in the presentcontext, corresponds to the above-described term “percent (%) identity”,taking into consideration the conservative amino acid substitutions, inaddition to the identical amino acids, when calculating the percentage.

[0046] The term “homology” as used in the present context, in turn,indicates evolutionary relationship. Two homologous proteins havedeveloped from a shared precursor sequence. The term does notnecessarily have anything to do with identity or similarity, apart fromthe fact that homologous sequences usually have a higher degree ofsimilarity (or occupy more identical positions in an alignment) thannon-homologous sequences.

[0047] The nucleic acids according to the invention preferably take theform of DNA or DNA fragments which correspond to genomic insect DNA, thenucleic acids preferably originating from dipterans, especiallypreferably from Drosophilidae.

[0048] The nucleic acids according to the invention especiallypreferably take the form of DNA or DNA fragments which correspond togenomic DNA of Myzus persicae or Drosophila melanogaster.

[0049] Very especially preferably, the nucleic acids according to theinvention encompass a sequence selected from

[0050] a) the sequence as shown in SEQ ID NO: 1,

[0051] b) the sequence as shown in Accession Number AAF59156,

[0052] c) part-sequences of the sequences defined under a) or b) whichare at least 14 base pairs in length,

[0053] d) sequences which hybridize with the sequences defined under a)or b) at a hybridization temperature of from 37° C. to 50° C.,

[0054] e) sequences which have at least 60%, preferably 80%, especiallypreferably 90% and very especially preferably 95% identity with thesequences defined under a) and b),

[0055] f) sequences which are complementary to the sequences definedunder a) to e), and

[0056] g) sequences which, owing to the degeneracy of the genetic code,encode the same amino acid sequence as the sequences defined under a) toe).

[0057] A very especially preferred embodiment of the nucleic acids to beused in accordance with the invention is a cDNA molecule with thesequence encoding the Myzus persicae ACCase and the sequence as shown inSEQ ID NO: 1 encoding the Drosophila melanogaster ACCase.

[0058] Based on the genetic code, the nucleic acid sequence encoding theMyzus persicae ACCase can be deduced from the amino acid sequence whichcan be isolated as described in Example 3 and defined by means ofsequencing.

[0059] Owing to the degeneracy of the genetic code, it is important touse the deduced nucleic acid sequence for verifying the nucleic acidsequence which is actually present in Myzus persicae and, ifappropriate, correcting the deduced sequence, as far as this makessense.

[0060] Isolating or verifying the genomic M. persicae sequence can beeffected for example by using the primers which are derived from thededuced nucleic acid sequence and which can be utilized in PCR reactionsfor amplifying the target sequence by methods known to the skilledworker.

[0061] The present invention also relates to the polypeptides which areencoded by the nucleic acids according to the invention.

[0062] The term “to hybridize” as used in the present context describesthe process in which a single-stranded nucleic acid molecule undergoesbase pairing with a complementary strand. For example, starting from thesequence information which is mentioned herein or which can be deduced,DNA fragments can be isolated, in this manner, from insects other thanDrosophila melanogaster which encode ACCases with the same or similarproperties of one of the ACCases according to the invention.

[0063] Hybridization conditions are calculated approximately by thefollowing formula:

[0064] Melting temperature Tm=81.5° C.+16.6 (log[c(Na⁺)])+0.41(%G+C)−500/n (Lottspeich & Zorbas 1998).

[0065] In this formula, c is the concentration and n the length of thehybridizing sequence segment in base pairs. For a sequence >100 bp, theterm 500/n is dropped. The highest stringency involves washing at atemperature of 5-15° C. below Tm and an ionic strength of 15 mM Na⁺(corresponds to 0.1×SSC). If an RNA sample is used for hybridization,the melting point is 10-15° C. higher.

[0066] Preferred hybridization conditions are stated hereinbelow:

[0067] Hybridization solution: DIG Easy Hyb (Roche, ZZ), hybridizationtemperature: 37° C. to 50° C., preferably 42° C. (DNA-DNA), 50° C.(DNA-RNA).

[0068] 1. Wash step: 2×SSC, 0.1% SDS 2×5 min at room temperature;

[0069] 2. Wash step: 1×SSC, 0.1% SDS 2×15 min at 50° C.; preferably0.5×SSC, 0.1% SDS 2×15 min at 65° C.; especially preferably 0.2×SSC,2×15 min at 68° C.

[0070] The degree of identity of the nucleic acids is preferablydetermined with the aid of the program NCBI BLASTN Version 2.0.4.(Altschul et al. 1997).

[0071] The term “regulatory regions” as used in the present contextrefers to untranslated regions of the gene in question, such aspromoters, enhancers, repressor or activator binding sites ortermination sequences, which interact with cellular proteins, thusgoverning transcription.

[0072] The present invention also relates to DNA constructs whichencompass a nucleic acid to be used in accordance with the invention anda heterologous promoter.

[0073] The present invention furthermore relates to the use of such DNAconstructs for identifying ACCase modulators.

[0074] The term “heterologous promoter” as used in the present contextrefers to a promoter with properties other than the promoter whichcontrols the expression of the gene in question in the originalorganism.

[0075] The choice of heterologous promoters depends on whetherprokaryotic or eukaryotic cells or cell-free systems are used forexpression. Examples of heterologous promoters are the SV40, theadenovirus or the cytomegalovirus early or late promoter, the lacsystem, the trp system, the main operator and promoter regions of phagelambda, the control regions of the fd coat protein, the3-phosphoglycerate kinase promoter, the acid phosphatase promoter, theBaculovirus immediate early promoter and the yeast α-mating factorpromoter.

[0076] The invention furthermore relates to vectors comprising a nucleicacid according to the invention or a DNA construct according to theinvention. Vectors which can be used are all those plasmids, phasmids,cosmids, YACs or artificial chromosomes which are used inmolecular-biological laboratories.

[0077] The invention furthermore relates to the use of vectorscomprising a nucleic acid to be used in accordance with the invention ora DNA construct to be used in accordance with the invention in methodsfor identifying ACCase modulators.

[0078] Vectors which can be used are all those phages, plasmids,phagmids, phasmids, cosmids, YACs, BACs, artificial chromosomes orparticles suitable for particle bombardment which are used inmolecular-biological laboratories.

[0079] The present invention also relates to host cells comprising anucleic acid to be used in accordance with the invention, a DNAconstruct to be used in accordance with the invention or a vector to beused in accordance with the invention.

[0080] The present invention also relates to the use of such host cellsfor identifying ACCase modulators.

[0081] The term “host cell” as used in the present context refers tocells which do not naturally contain the nucleic acids to be used inaccordance with the invention.

[0082] Suitable host cells are prokaryotic cells, such as bacteria ofthe genera Bacillus, Pseudomonas, Streptomyces, Streptococcus,Staphylococcus, preferably E. coli, but also eukaryotic cells such asyeasts, mammalian cells, amphibian cells, insect cells or plant cells.Preferred eukaryotic host cells are HEK 293 cells, Schneider S2 cells,Spodoptera Sf9 cells, Kc cells, CHO cells, COS1 cells, COS7 cells, HeLacells, C127 cells, 3T3 cells or BHK cells, in particular Xenopusoocytes.

[0083] The term “polypeptides” as used in the present context refers notonly to short amino acid chains which are generally referred to aspeptides, oligopeptides or oligomers, but also to longer amino acidchains which are normally referred to as proteins. It encompasses aminoacid chains which can be modified either by natural processes, such aspost-translational processing, or by chemical prior-art methods. Suchmodifications may occur at various sites and repeatedly in apolypeptide, such as, for example, on the peptide backbone, on the aminoacid side chain, on the amino and/or the carboxyl terminus. For example,they encompass acetylations, acylations, ADP ribosylations, amidations,covalent linkages to flavins, hem moieties, nucleotides or nucleotidederivatives, lipids or lipid derivatives or phosphatidylinositol,cyclizations, disulfide bridge formations, demethylations, cystineformations, formylations, gamma-carboxylations, glycosylations,hydroxylations, iodinations, methylations, myristoylations, oxidations,proteolytic processings, phosphorylations, selenoylations andtRNA-mediated amino acid additions.

[0084] The polypeptides according to the invention may exist in the formof “mature” proteins or as parts of larger proteins, for example asfusion proteins. They can furthermore exhibit secretion or leadersequences, pro-sequences, sequences which allow simple purification,such as polyhistidine residues, or additional stabilizing amino acids.The proteins according to the invention may also exist in the form inwhich they are naturally present in their organism of origin, from whichthey can be obtained directly, for example.

[0085] The term “complete ACCase” as used in the present contextdescribes an ACCase which is encoded by a complete coding region of atranscription unit starting with the ATG start codon and comprising allinformation-bearing exon regions of the ACCase-encoding gene which ispresent in the organism of origin, and signals required for correcttranscriptional termination.

[0086] The term “gene” as used in the present context refers to asegment from the genome of a cell which is responsible for the synthesisof a polypeptide chain.

[0087] The polypeptides according to the invention need not be completeACCases, but may also take the form of fragments thereof, as long asthey show at least the biological activity of the complete ACCase.Polypeptides from insects which exert the same type of biologicalactivity as a Myzus persicae ACCase or Drosophila melanogaster ACCaseare still considered as being in accordance with the invention. In thiscontext, the polypeptides according to the invention need not correspondfully to the Myzus persicae or Drosophila melanogaster ACCases withregard to their sequence or catalytic activity. Polypeptides which arealso considered as polypeptides according to the invention are thosewhich are homologous to the ACCase from, for example, the followinginsects or to fragments thereof which retain the biological activity ofACCase:

[0088] From the order of the Isopoda, for example, Oniscus asellus,Armadillidium vulgare, Porcellio scaber.

[0089] From the order of the Diplopoda, for example, Blaniulusguttulatus.

[0090] From the order of the Chilopoda, for example, Geophiluscarpophagus, Scutigera spp.

[0091] From the order of the Symphyla, for example, Scutigerellaimmaculata.

[0092] From the order of the Thysanura, for example, Lepisma saccharina.

[0093] From the order of the Collembola, for example, Onychiurusarmatus.

[0094] From the order of the Orthoptera, for example, Acheta domesticus,Gryllotalpa spp., Locusta migratoria migratorioides, Melanoplus spp.,Schistocerca gregaria.

[0095] From the order of the Blattaria, for example, Blatta orientalis,Periplaneta americana, Leucophaea maderae, Blattella germanica.

[0096] From the order of the Dermaptera, for example, Forficulaauricularia.

[0097] From the order of the Isoptera, for example, Reticulitermes spp.

[0098] From the order of the Phthiraptera, for example, Pediculushumanus corporis, Haematopinus spp., Linognathus spp., Trichodectesspp., Damalinia spp.

[0099] From the order of the Thysanoptera, for example, Hercinothripsfemoralis, Thrips tabaci, Thrips palmi, Frankliniella accidentalis.

[0100] From the order of the Heteroptera, for example, Eurygaster spp.,Dysdercus intermedius, Piesma quadrata, Cimex lectularius, Rhodniusprolixus, Triatoma spp.

[0101] From the order of the Homoptera, for example, Aleurodesbrassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis gossypii,Brevicoryne brassicae, Cryptomyzus ribis, Aphis fabae, Aphis pomi,Eriosoma lanigerum, Hyalopterus arundinis, Phylloxera vastatrix,Pemphigus spp., Macrosiphum avenae, Myzus spp., Phorodon humuli,Rhopalosiphum padi, Empoasca spp., Euscelis bilobatus, Nephotettixcincticeps, Lecanium corni, Saissetia oleae, Laodelphax striatellus,Nilaparvata lugens, Aonidiella aurantii, Aspidiotus hederae,Pseudococcus spp., Psylla spp.

[0102] From the order of the Lepidoptera, for example, Pectinophoragossypiella, Bupalus piniarius, Cheimatobia brumata, Lithocolletisblancardella, Hyponomeuta padella, Plutella xylostella, Malacosomaneustria, Euproctis chrysorrhoea, Lymantria spp., Bucculatrixthurberiella, Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltiaspp., Earias insulana, Heliothis spp., Mamestra brassicae, Panolisflammea, Spodoptera spp., Trichoplusia ni, Carpocapsa pomonella, Pierisspp., Chilo spp., Pyrausta nubilalis, Ephestia kuehniella, Galleriamellonella, Tineola bisselliella, Tinea pellionella, Hofmannophilapseudospretella, Cacoecia podana, Capua reticulana, Choristoneurafumiferana, Clysia ambiguella, Homona magnanima, Tortrix viridana,Cnaphalocerus spp., Oulema oryzae.

[0103] From the order of the Coleoptera, for example, Anobium punctatum,Rhizopertha dominica, Bruchidius obtectus, Acanthoscelides obtectus,Hylotrupes bajulus, Agelastica alni, Leptinotarsa decemlineata, Phaedoncochleariae, Diabrotica spp., Psylliodes chrysocephala, Epilachnavarivestis, Atomaria spp., Oryzaephilus surinamensis, Anthonomus spp.,Sitophilus spp., Otiorrhynchus sulcatus, Cosmopolites sordidus,Ceuthorrhynchus assimilis, Hypera postica, Dermestes spp., Trogodermaspp., Anthrenus spp., Attagenus spp., Lyctus spp., Meligethes aeneus,Ptinus spp., Niptus hololeucus, Gibbium psylloides, Tribolium spp.,Tenebrio molitor, Agriotes spp., Conoderus spp., Melolontha melolontha,Amphimallon solstitialis, Costelytra zealandica, Lissorhoptrusoryzophilus.

[0104] From the order of the Hymenoptera, for example, Diprion spp.,Hoplocampa spp., Lasius spp., Monomorium pharaonis, Vespa spp.

[0105] From the order of the Diptera, for example, Aedes spp., Anophelesspp., Culex spp., Drosophila melanogaster, Musca spp., Fannia spp.,Calliphora erythrocephala, Lucilia spp., Chrysomyia spp., Cuterebraspp., Gastrophilus spp., Hyppobosca spp., Stomoxys spp., Oestrus spp.,Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus, Oscinellafrit, Phorbia spp., Pegomyia hyoscyami, Ceratitis capitata, Dacus oleae,Tipula paludosa, Hylemyia spp., Liriomyza spp.

[0106] From the order of the Siphonaptera, for example, Xenopsyllacheopis, Ceratophyllus spp.

[0107] In comparison with the corresponding regions of naturallyoccurring ACCases, the polypeptides according to the invention can havedeletions or amino acid substitutions as long as they still exert atleast one biological activity of the complete ACCases. Conservativesubstitutions are preferred. Such conservative substitutions encompassvariations, one amino acid being replaced by another amino acid fromamong the following group:

[0108] 1. small aliphatic residues, unpolar residues or residues oflittle polarity: Ala, Ser, Thr, Pro and Gly;

[0109] 2. polar, negatively charged residues and their amides: Asp, Asn,Glu and Gln;

[0110] 3. polar, positively charged residues: His, Arg and Lys;

[0111] 4. large aliphatic unpolar residues: Met, Leu, Ile, Val and Cys;and

[0112] 5. aromatic residues: Phe, Tyr and Trp.

[0113] Preferred conservative substitutions can be seen from thefollowing list: Original residue Substitution Ala Gly, Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala, Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Tyr, Ile Phe Met, Leu,Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0114] The present invention therefore also relates to polypeptideswhich exert at least the biological activity of an ACCase and whichcomprise an amino acid sequence with at least 60% identity, preferably80%, especially preferably 90% identity and very especially preferably95% identity with the Myzus persicae or the Drosophila melanogastersequence encoded by the nucleic acid as shown in SEQ ID NO: 1, and theiruse for identifying ACCase modulators.

[0115] The term “biological activity of an ACCase” as used in thepresent context refers to the ability to catalyze the biotin-dependentcarboxylation of acetyl-CoA. In this context, all three enzymefunctions, i.e. the ATP-dependent elimination of a CO₂ group frombicarbonate, the biotin carrier function and the carboxylation ofacetyl-CoA, or else only one or two of these reactions may beencompassed.

[0116] The nucleic acids according to the invention can be prepared inthe customary manner. For example, the nucleic acid molecules in theirentirety can be synthesized chemically, or else short sections of thenucleic acids according to the invention can be synthesized chemically,and such oligonucleotides can be radiolabeled or labeled with afluorescent dye. The labeled oligonucleotides can also be used forscreening cDNA libraries generated starting from insect mRNA. Cloneswith which the labeled oligonucleotides hybridize are chosen forisolating the DNA fragments in question. After characterization of theDNA which has been isolated, the nucleic acids according to theinvention are obtained in a simple manner.

[0117] As an alternative, the nucleic acids according to the inventioncan be generated by means of PCR methods using chemically synthesizedoligonucleotides.

[0118] The term “oligonucleotide(s)” as used in the present contextrefers to DNA molecules composed of 10 to 50 nucleotides, preferably 15to 30 nucleotides. They are synthesized chemically and can be used asprobes.

[0119] Moreover, host cells comprising the nucleic acids according tothe invention may be cultured under suitable conditions in order toprepare the polypeptides according to the invention, in particular thepolypeptide encoded by the nucleic acid sequence as shown in SEQ IDNO: 1. Thereafter, the desired polypeptides can be isolated in thecustomary manner from the cells or from the culture medium. As analternative, the polypeptides may be generated in in-vitro systems.

[0120] To prepare the Myzus persicae ACCase according to the invention,a procedure may be followed in which larvae or adults are homogenizedwith a pestle and mortar. To this end, they may previously be frozenrapidly, for example in liquid nitrogen. The homogenate is taken up in asuitable buffer. An example of the preparation of a polypeptideaccording to the invention is given in Example 3.

[0121] One possible ACCase purification method is based on preparativeelectrophoresis, FPLC, HPLC (for example, using gel filtration columns,reversed-phase columns or mildly hydrophobic columns), gel filtration,differential precipitation, ion-exchange chromatography or affinitychromatography.

[0122] A rapid method of isolating the polypeptides according to theinvention which are synthesized by host cells using a nucleic acidaccording to the invention starts with expressing a fusion protein,where the fusion moiety may be purified in a simple manner by affinitypurification. For example, the fusion moiety may be glutathioneS-transferase. The fusion protein can then be purified on a glutathioneaffinity column. The fusion moiety can be removed by partial proteolyticcleavage, for example at linkers between the fusion moiety and thepolypeptide according to the invention which is to be purified. Thelinker can be designed in such a way that it includes target aminoacids, such as arginine and lysine residues, which define sites fortrypsin cleavage. Standard cloning methods using oligonucleotides may beemployed for generating such linkers.

[0123] Other purification methods which are possible are based, in turn,on preparative electrophoresis, FPLC, HPLC (for example, using gelfiltration columns, reversed-phase columns or mildly hydrophobiccolumns), gel filtration, differential precipitation, ion-exchangechromatography or affinity chromatography.

[0124] The terms “isolation or purification” as used in the presentcontext mean that the polypeptides according to the invention areseparated from other proteins or other macromolecules of the cell or ofthe tissue. The protein content of the composition containing thepolypeptides according to the invention is preferably at least 10 times,especially preferably at least 100 times, higher than in a host cellpreparation.

[0125] The polypeptides according to the invention may also be subjectedto affinity purification without fusion moiety with the aid ofantibodies which bind to the polypeptides.

[0126] The present invention also relates to methods of finding chemicalcompounds which bind to ACCase and/or modify its properties. Owing tothe important function of ACCase, modulators which affect the activityconstitute novel insecticidal and/or, if appropriate, acaricidal activeingredients. Modulators may be agonists or antagonists, or inhibitors oractivators.

[0127] Owing to the property of acting as inhibitors of insect ACCase,the abovementioned cyclic 1,3-dicarbonyl compounds of the formula (I)and their enols may also be used as optionally labeled competitors inmethods for finding insect ACCase inhibitors which need not belong tothis group of compounds.

[0128] The term “competitor” as used in the present context refers tothe property of the compounds of competing with other compounds, ifappropriate compounds yet to be identified, for binding to ACCase and ofdisplacing the former from the enzyme, or of being displaced thereby.

[0129] The term “agonist” as used in the present context refers to amolecule which accelerates or increases the ACCase activity.

[0130] The term “antagonist” as used in the present context refers to amolecule which slows down or prevents the ACCase activity.

[0131] The term “modulator” as used in the present context is a generalterm for agonist or antagonist. Modulators may be small organo-chemicalmolecules, peptides or antibodies which bind to the polypeptidesaccording to the invention and/or modify their properties, for exampletheir enzymatic activity. Moreover, modulators can be smallorgano-chemical molecules, peptides or antibodies which bind to amolecule which, in turn, binds to the polypeptides according to theinvention and/or influences their biological activity. Modulators can benatural substrates and ligands, or structural or functional mimetics ofthese.

[0132] The modulators preferably take the form of small organo-chemicalcompounds.

[0133] It was demonstrated for the first time within the scope of thepresent invention that compounds or modulators which act on ACCase arecapable of modifying the cellular processes in a manner which leads tothe death of the insects treated therewith.

[0134] The present invention therefore also relates to modulators ofinsect ACCases which are found with the aid of a method of identifyingACCase modulators, which method is described in the present application.

[0135] The present invention furthermore encompasses methods of findingchemical compounds which modify the expression of the polypeptidesaccording to the invention. Such “expression modulators” too may benovel insecticidal active compounds. Expression modulators can be smallorgano-chemical molecules, peptides or antibodies which bind to theregulatory regions of the nucleic acids encoding the polypeptidesaccording to the invention. Moreover, expression modulators may be smallorgano-chemical molecules, peptides or antibodies which bind to amolecule which, in turn, binds to regulatory regions of the nucleicacids encoding the polypeptides according to the invention, thusinfluencing their expression. Expression modulators may also beantisense molecules.

[0136] The present invention likewise relates to the use of modulatorsof the polypeptides according to the invention or of expressionmodulators as insecticides or acaricides.

[0137] The present invention likewise relates to ACCase expressionmodulators which are found with the aid of the above-described methodfor finding expression modulators.

[0138] The methods according to the invention include high-throughputscreening (HTS) and ultra-high-throughput screening (UHTS). Both hostcells and cell-free preparations which comprise the nucleic acidsaccording to the invention and/or the polypeptides according to theinvention may be used.

[0139] One way of finding modulators is the incubation of a syntheticreaction mix (for example products of the in-vitro transcription) or acellular component, such as a membrane, a compartment or any otherpreparation comprising the polypeptides according to the invention,together with a labeled substrate or ligand of the polypeptides in thepresence and absence of a candidate molecule, which may take the form ofan agonist or antagonist. The ability of the candidate molecule toincrease or to inhibit the activity of polypeptides according to theinvention can be seen from an increased or reduced binding of thelabeled ligand or from an increased or reduced conversion rate of thelabeled substrate. Molecules which bind well and which lead to increasedactivity of the polypeptides according to the invention are agonists.Molecules which bind well and which inhibit the biological activity ofthe polypeptides according to the invention are good antagonists. Theymay also take the form of inhibitors of the abovementioned class ofinsecticidal substances, but entirely novel classes of substances toomay show this modulatory activity.

[0140] Modulators which reduce the activity of a polypeptide accordingto the invention or the expression of mRNA encoding ACCase according tothe invention and/or polypeptides by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 95% are suitable for use asinsecticides or for being developed further to give insecticides. Suchcandidate molecules are then checked in further tests for toxicity tovertebrate species, such as, for example, mammals, and for theirbioavailability.

[0141] Detection of the biological activity of the polypeptidesaccording to the invention can be improved by what is known as areporter system. In this aspect, reporter systems comprise, but are notrestricted to, colorimetrically labeled or radiolabeled substrates whichare converted into a product, or a reporter gene which responds tochanges in the activity or the expression of the polypeptides accordingto the invention, or other known binding assays.

[0142] The activity of a large number of proteins, for exampletransmembrane proteins, can be measured advantageously in a furthermanner. The functional heterologous expression of such proteins in E.coli is frequently difficult or impossible. In this case, thecatalytically active part of the protein can be separated by means ofsuitable cloning methods (for example using suitable PCR strategies), sothat the gene product is a soluble protein, or a protein with bettersolubility, and can be purified readily. A wide range of possiblemethods is available for measuring the activity of soluble proteins. Aparticularly sensitive measurement can be carried out for example bymeans of fluorescence polarization using a fluorescently labeled ligandor substrate.

[0143] A further example of a method by means of which modulators of thepolypeptides according to the invention can be found is a displacementassay in which the polypeptides according to the invention and apotential modulator are combined, under suitable conditions, with amolecule which is known to bind to the polypeptides according to theinvention, such as a natural substrate or ligands or a substrate orligand mimetic. An example is the abovementioned compounds of theformula (I). The polypeptides according to the invention can themselvesbe labeled, for example radiolabeled or calorimetrically labeled, sothat the number of polypeptides which are bound to a ligand or whichhave undergone a conversion can be determined accurately. The efficacyof an agonist or antagonist can be determined in this manner.

[0144] Potentially insecticidal compounds which are found in one of themethods according to the invention with the aid of the nucleic acidsand/or polypeptides according to the invention can be administered tothe insects in a variety of ways, for example orally (see also Example4), topically or by injection. Insecticides are frequently hydrophobicmolecules and must, in such a case, usually be dissolved in organicsolvents which are also capable of evaporation (for example methanol oracetone) or which are added in minor concentrations in order tofacilitate uptake (ethanol, dimethyl sulfoxide).

[0145] The first step in insect experiments is, as a rule, thedetermination of the MLD (minimal lethal dose) following chronicexposure of the insects. Usually, the compounds are diluted and added tothe feed of embryos and larvae aged 0-48 hours. In addition to the MID,this procedure also determines the percentage of eggs from which larvaestill hatch, and the behavior of the larvae (movement, uptake of feed),the number of larvae which still pupate and the number of adults whichthey produce. Moreover, the larvae can be studied for morphologicaldefects. After the MLD has been determined, the acute and chronic dosemay be determined.

[0146] In a typical acute test, the compounds are added to the food ofembryos, larvae or adults, and the insects are checked after 2 hours orfollowing incubation overnight. In the case of embryos, the number ofembryos with development defects and the percentage which survives intoadulthood are determined.

[0147] In larvae, parameters which are studied are, for example,abnormal behavior, impaired movement or ecdysis. In adult animals,defects regarding the quantity or activity of enzymes, abnormal behaviorand/or impaired fertility are observed.

[0148] To carry out tests for determining the chronic toxicity, theadults are placed into dishes containing the compound in question, forexample for 48 hours, and they are then transferred into a cleancontainer and the fertility of the animals or the amount of activity ofa certain enzyme or the death of the insects are observed.

[0149] The examples which follow demonstrate that, surprisingly, ACCaseis an essential enzyme in insects; moreover, they demonstrate that theenzyme is a suitable target protein for identifying insecticides, thatit can be used in methods for identifying insecticidally activecompounds and that the ACCase modulators which are identified insuitable methods can be used as insecticides. To make possible the useof ACCase, obtaining this enzyme from Myzus persicae is described by wayof example, and, finally, the applicability of the present invention inthe search for insecticidally active compounds is demonstrated.

EXAMPLES Example 1 Preparation of an ACCase Enzyme Preparation fromMyzus persicae

[0150]Myzus persicae larvae or adults are weighed and homogenized inthree times the amount of extraction buffer using a pestle and mortar.The extract is subsequently centrifuged twice for 10 minutes at 10 000g. The supernatant contains ACCase and is used in the enzyme assay foridentifying inhibitors.

[0151] The following buffer is preferably used as extraction buffer:0.25 M sucrose, 15 mM tris/HCl pH 7.4, 4 mM EDTA, 10 mM potassiumcitrate (all chemicals from Sigma, St. Louis).

Example 2 Method for Finding Modulators

[0152] The ACCase enzyme preparation was used in a biochemical assay asdescribed hereinbelow for finding ACCase modulators: first, an aliquotof the enzyme preparation of Example 1 was mixed with the reactionbuffer and the radiolabeled substrate and the mixture was incubated. Todetect the incorporation of CO₂, fuming HCl was pipetted in, and analiquot of the reaction mixture was added dropwise to Watman filterpaper and dried. The dried filter paper was transferred intoscintillation tubes together with scintillation liquid. The measurementwas performed in a scintillation counter (Beckman Instruments,Fullerton, USA). To screen for modulators, the test compounds weredissolved in DMSO and added to the reaction mixture together with theenzyme preparation before the first incubation step. The effect of themodulators was determined in comparison with the ACCase activity in thereaction mixture with solvent, but without modulator. FIG. 2a shows theinhibition of the Myzus persicae ACCase at different active ingredientconcentrations. FIG. 2b shows the inhibition of the Myzus persicaeACCase by a range of active ingredients.

[0153] The reaction buffer used was 50 mM Tris/HCl pH 7.4, 15 mM MgCl₂,2.5 mM ATP, 1 μg/μl bovine serum albumin, 10 mM potassium citrate, 84 mMsodium bicarbonate (all chemicals from Sigma, St. Louis).

[0154] 4 mM ¹⁴C sodium bicarbonate was used as the radiolabeledsubstrate.

Example 3 Measuring the ACCase Activity for Finding ACCase Inhibitors

[0155] A part-step of the ACCase-catalyzed reaction is the fixation ofthe carbonate group at the cofactor biotin. This fixation takes placewith the cleavage of ATP: ACCase-biotin+HCO₃ ⁻+ATP→ACCase-biotin-CO₂⁻+ADP+P_(i). To determine the ACCase activity, the phosphate beingliberated is detected with the commercially available malachite greenreagent. Since this takes the form of a general (unspecific) detectionof phosphate, all of the materials and reagents used must be free fromphosphate. The ACCase must be purified from other ATP-cleaving enzymesfor this detection reaction.

[0156] (a) ACCase Preparation

[0157] Whole insects, or tissues/organs obtained from them, arecomminuted in the homogenization buffer (250 mM sucrose, 50 mM tris-HCl,pH 7.4, 2 mM EDTA, 10 mM sodium citrate, proteinase inhibitor mix (SigmaP-8340), for example by comminuting in a pestle and mortar or using ahomogenizer rod). The homogenate is then centrifuged to obtain a clearmaterial. The supernatant, which contains the ACCase, is then subjectedto a buffer exchange with the running buffer (50 mM Tris-HCl pH 7.4, 150mM NaCl, 1 mM EDTA, 1 mM DTT, 0.02% NaN₃, 10% glycerol) for the furthersteps, using a 4 PD-10 column (Pharmacia Corporation, Peapack, N.J.,USA). The crude extract is subsequently separated in a Sephacryl 26/60S-300 column (Pharmacia Corporation, Peapack, N.J., USA) by FPLC(Pharmacia Corporation, Peapack, N.J., USA). The fractions obtained areassayed for ACCase activity as described under (b). The fractions withthe highest specific ACCase activities are combined and constitute thestarting materials for the inhibitor measurements described under (c)(hereinbelow referred to as ACCase solution).

[0158] (b) Activity Assay

[0159] An aliquot of the ACCase solution described under (a) is mixedwith the reaction buffer (50 mM Tris-HCl pH 7.4, 20 mM MgCl₂, 1 mg/mlbovine serum albumin, 20 mM sodium citrate, 5 mM NaHCO₃, 1 mM ATP, 200μM acetyl-CoA), and the mixture is incubated at physiologicaltemperatures (24° C. to 37° C.). After the desired reaction time haselapsed, 2 volumes of the staining reagent (BiomolGreen (Biomol ResearchLaboratories Inc., Plymouth Meeting, Pa., USA), prepared as specified bythe manufacturer) are added and incubation of the mixture is continuedas specified by the manufacturer until the detection reaction has becomeapparent. To determine the unspecific background activity, reactionmixtures without the substrate acetyl-CoA are also included in themeasurement, and the results are subtracted when calculating thespecific ACCase activity.

[0160] (c) Method for Identifying ACCase Inhibitors

[0161] To determine the inhibition of ACCase by test substances,aliquots of the ACCase solution of (a) are mixed with the reactionbuffer and the test inhibitors, and the mixture is incubated atphysiological temperatures (24° C. to 37° C.). After the desiredreaction time has elapsed, the detection reaction is carried out asdescribed under (b). A control measurement without addition of inhibitoris carried out in parallel. The ACCase inhibition is then calculated incomparison with this control.

[0162] Supplier: unless specified otherwise, all chemicals are fromSigma-Aldrich Co., St. Louis, USA.

Example 4 Impairment of Lipid Neogenesis by ACCase Inhibitors

[0163] Peach aphid Myzus persicae larvae or adults were fed for two dayswith nutrient solutions with and without one of the identified ACCaseinhibitors, using the sachet method (Nauen et al. 1996).

[0164] By way of example, the compound of the formula (I-A)

[0165] which can be referred to as4-hydroxy-8-methoxy-3-(2,3,4,6-tetramethylphenyl)-1-azaspiro[4,5]dec-32-en-2-one,was used in this experiment.

[0166] Thereafter, the animals were collected and homogenized in organicsolvent using a pestle and mortar. The organic phase was cleansed ofwater-soluble constituents by repeatedly extracting it by shaking withaqueous solution, and the solvent was then evaporated. The pellet whichremained was taken up in a little solvent and separated by thin-layerchromatography (TLC). The lipids which were separated were stained withamido black. ¹⁴C-acetate which was incorporated into the lipids wasdetected by autoradiography after an exposure time of 2-3 days. Toquantify the ¹⁴C-incorporation into lipids, the bands in question werescraped out after staining, dissolved, and the activity was determinedin a scintillation counter. FIG. 1a shows the lipid status after acetatefeeding without (lanes 1-3) and with the active ingredient of theformula (I-A) (lanes 4-6) with reference to a separated lipid extractfrom the peach aphid Myzus persicae. No significant differences in lipidcomposition and lipid content are observed. FIG. 1b shows anautoradiograph of the same TLC plate. While in lanes 1-3 those lipidsinto which radiolabeled acetate was incorporated during de-novosynthesis in the control aphids are discernible owing to the blackcolor, no labeled lipids are present in the peach aphids treated withthe active ingredient. Thus, no de novo lipid synthesis from acetate hastaken place. FIG. 1c again shows, in the form of a diagram, how muchacetate was still incorporated during de-novo synthesis in the presenceof an ACCase inhibitor (0.01 ppm to 100 ppm) in comparison with thecontrol without ACCase inhibitor. It can be seen clearly that increasinginhibitor concentrations halt the de-novo lipid biosynthesis.

[0167] The mobile phase used for the TLC chromatography was n-hexane:diethyl ether: glacial acetic acid (60:45:1).

DESCRIPTION OF THE FIGURES

[0168]FIG. 1a)

[0169] Separated lipid extract from peach aphids Myzus persicaefollowing acetate feeding without (lanes 1-3) and with an ACCaseinhibitor (lanes 4-6). The lipids which were separated were stained withamido black.

[0170]FIG. 1b)

[0171] Autoradiograph of the TLC plate shown in FIG. 1a). ¹⁴C-acetatewas detected after an exposure time of 2-3 days by means of an image.While those lipids into which radiolabeled acetate had been incorporatedin de-novo synthesis in the control aphids are discernible in lanes 1-3owing to their black color, no labeled lipids are present in the peachaphids treated with active ingredient.

[0172]FIG. 1c)

[0173] Comparison of the incorporation of ¹⁴C-acetate in the absence andpresence of ACCase inhibitors (0.01 to 100 ppm) during de-novo lipidsynthesis.

[0174]FIG. 2a)

[0175] Inhibition of Myzus persicae ACCase at different activeingredient concentrations. Solv=solvent (control). Comp=compound (activeingredient).

[0176]FIG. 2b)

[0177] Inhibition of Myzus persicae ACCase by different activeingredients. Comp=compound.

[0178]FIG. 3)

[0179] Inhibition of the ACCase from peach aphids Myzus persicae by twodifferent substances A and B. The figure shows the respective ACCaseactivity at different inhibitor concentrations in comparison with thecontrol (mixture without inhibitor not shown).

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1 2 1 7047 DNA Drosophila melanogaster CDS (1)..(7047) 1 atg ttg aag cgtcgc gcc agc aag cgt ttc gta ctt gtt gag tcc ggt 48 Met Leu Lys Arg ArgAla Ser Lys Arg Phe Val Leu Val Glu Ser Gly 1 5 10 15 gaa gat aat gccaac ggc tcc ggc tcg gcc tcg ggc tct ggc tcg gga 96 Glu Asp Asn Ala AsnGly Ser Gly Ser Ala Ser Gly Ser Gly Ser Gly 20 25 30 tca gga gtg gga acggcg gtt ata ccc caa ttt gtg gct gtg gat tgc 144 Ser Gly Val Gly Thr AlaVal Ile Pro Gln Phe Val Ala Val Asp Cys 35 40 45 ggg cag aac gag agc aacaac aac cat gtc ggc gag atg agt gcc agc 192 Gly Gln Asn Glu Ser Asn AsnAsn His Val Gly Glu Met Ser Ala Ser 50 55 60 atc agc aat cac aat agc tccaac aac cag tcg tcg cca tcg ctg ctc 240 Ile Ser Asn His Asn Ser Ser AsnAsn Gln Ser Ser Pro Ser Leu Leu 65 70 75 80 agt gtg ccc gtg gtg gga accctc aag ccg agt atg tcg cgt ggc aca 288 Ser Val Pro Val Val Gly Thr LeuLys Pro Ser Met Ser Arg Gly Thr 85 90 95 ggg ctg ggc cag gac cgg cac caggat cgc gac ttc cac atc gca acc 336 Gly Leu Gly Gln Asp Arg His Gln AspArg Asp Phe His Ile Ala Thr 100 105 110 acc gag gag ttc gtg aag cgc tttggc ggc acc cga gtg atc aac aag 384 Thr Glu Glu Phe Val Lys Arg Phe GlyGly Thr Arg Val Ile Asn Lys 115 120 125 gtc ctg att gcc aac aac ggt atcgcg gcc gtc aag tgc atg cga tcc 432 Val Leu Ile Ala Asn Asn Gly Ile AlaAla Val Lys Cys Met Arg Ser 130 135 140 atc cgg aga tgg gcg tac gaa atgttt aag aac gag cgg gcc att agg 480 Ile Arg Arg Trp Ala Tyr Glu Met PheLys Asn Glu Arg Ala Ile Arg 145 150 155 160 ttt gtg gtg atg gtc act ccggag gat cta aag gcg aat gcc gaa tac 528 Phe Val Val Met Val Thr Pro GluAsp Leu Lys Ala Asn Ala Glu Tyr 165 170 175 atc aag atg gcg gat cac tatgtg ccc gtg ccc ggc gga tcg aac aac 576 Ile Lys Met Ala Asp His Tyr ValPro Val Pro Gly Gly Ser Asn Asn 180 185 190 aac aac tac gcc aat gtc gagctc atc gtg gac att gct ctt cgc acc 624 Asn Asn Tyr Ala Asn Val Glu LeuIle Val Asp Ile Ala Leu Arg Thr 195 200 205 caa gtg cag gcc gtg tgg gctggt tgg ggt cat gcc tcc gag aac ccg 672 Gln Val Gln Ala Val Trp Ala GlyTrp Gly His Ala Ser Glu Asn Pro 210 215 220 aag ctg ccg gag ctg ctt cacaaa gag ggt ctg gtg ttc ctt ggc cct 720 Lys Leu Pro Glu Leu Leu His LysGlu Gly Leu Val Phe Leu Gly Pro 225 230 235 240 ccg gaa cgt gcc atg tgggcg ctg ggc gac aag gtg gcc tcc tct att 768 Pro Glu Arg Ala Met Trp AlaLeu Gly Asp Lys Val Ala Ser Ser Ile 245 250 255 gtg gcc caa acg gcc gagatt ccc acc ctg ccg tgg tcc ggt tcg gac 816 Val Ala Gln Thr Ala Glu IlePro Thr Leu Pro Trp Ser Gly Ser Asp 260 265 270 ctg aag gcc cag tac agtggc aaa aag atc aag att tcc agt gag ctc 864 Leu Lys Ala Gln Tyr Ser GlyLys Lys Ile Lys Ile Ser Ser Glu Leu 275 280 285 ttc gcc cga ggt tgt gtgacc aat gtg gaa cag ggt ctg gcc gca gtt 912 Phe Ala Arg Gly Cys Val ThrAsn Val Glu Gln Gly Leu Ala Ala Val 290 295 300 aac aag att ggc ttc cccgta atg atc aag gcc tcg gaa gga ggt ggt 960 Asn Lys Ile Gly Phe Pro ValMet Ile Lys Ala Ser Glu Gly Gly Gly 305 310 315 320 ggc aag ggt att cgccgc gtg gac acc act gag gag ttc ccc ggc ctg 1008 Gly Lys Gly Ile Arg ArgVal Asp Thr Thr Glu Glu Phe Pro Gly Leu 325 330 335 ttc cgc cag gtt caagct gag gtg ccc ggc tca ccg att ttc gtg atg 1056 Phe Arg Gln Val Gln AlaGlu Val Pro Gly Ser Pro Ile Phe Val Met 340 345 350 aag ctg gcc cgc ggagct cgc cac ttg gag gtg caa ctg ttg gca gat 1104 Lys Leu Ala Arg Gly AlaArg His Leu Glu Val Gln Leu Leu Ala Asp 355 360 365 cag tac ggc aat gccatt agc ttg ttc ggc cgt gac tgc tcc atc cag 1152 Gln Tyr Gly Asn Ala IleSer Leu Phe Gly Arg Asp Cys Ser Ile Gln 370 375 380 cgt cgt cat cag aaaatt att gag gaa gct cct gcc atc gtg gcc cag 1200 Arg Arg His Gln Lys IleIle Glu Glu Ala Pro Ala Ile Val Ala Gln 385 390 395 400 cca gag gtg ttcgag gac atg gag aag gcc gcc gtg cgg ttg gcc aag 1248 Pro Glu Val Phe GluAsp Met Glu Lys Ala Ala Val Arg Leu Ala Lys 405 410 415 atg gtg ggt tacgtc agc gcg gga acc gtg gag tac cta tat gat ccg 1296 Met Val Gly Tyr ValSer Ala Gly Thr Val Glu Tyr Leu Tyr Asp Pro 420 425 430 gag ggt cgc tacttc ttc ctg gag ctg aac cca cgt ttg cag gtg gag 1344 Glu Gly Arg Tyr PhePhe Leu Glu Leu Asn Pro Arg Leu Gln Val Glu 435 440 445 cat ccg tgt acggag atg gtg gcc gat gta aat ctt cca gct gct cag 1392 His Pro Cys Thr GluMet Val Ala Asp Val Asn Leu Pro Ala Ala Gln 450 455 460 ctg cag att ggaatg gga att ccc ctt tac cgg ctc aag gac atc cgt 1440 Leu Gln Ile Gly MetGly Ile Pro Leu Tyr Arg Leu Lys Asp Ile Arg 465 470 475 480 ctg ctg tacgga gag tct ccc tgg ggc tcc tca gtc att gac ttc gaa 1488 Leu Leu Tyr GlyGlu Ser Pro Trp Gly Ser Ser Val Ile Asp Phe Glu 485 490 495 aat cca ccgaac aaa ccg cgt ccc tcc gga cat gtt atc gct gct cgt 1536 Asn Pro Pro AsnLys Pro Arg Pro Ser Gly His Val Ile Ala Ala Arg 500 505 510 atc acc tcagag aac ccc gac gag ggc ttt aag ccc agt tct gga acc 1584 Ile Thr Ser GluAsn Pro Asp Glu Gly Phe Lys Pro Ser Ser Gly Thr 515 520 525 gtt cag gagctt aac ttc cgg tcg agc aaa aat gtg tgg ggc tac ttc 1632 Val Gln Glu LeuAsn Phe Arg Ser Ser Lys Asn Val Trp Gly Tyr Phe 530 535 540 agt gtg gctgcc agt gga gga ttg cac gag ttc gcg gat tca cag ttt 1680 Ser Val Ala AlaSer Gly Gly Leu His Glu Phe Ala Asp Ser Gln Phe 545 550 555 560 ggg cattgt ttc tcc tgg ggc gag aac cgt caa cag gct cga gag aac 1728 Gly His CysPhe Ser Trp Gly Glu Asn Arg Gln Gln Ala Arg Glu Asn 565 570 575 ctg gtgatt gcc ctg aag gag ctg tca att cga ggt gat ttc cga acc 1776 Leu Val IleAla Leu Lys Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr 580 585 590 aca gtggaa tac ttg atc act ctg ctc gaa acg aat cgg ttc ctc gac 1824 Thr Val GluTyr Leu Ile Thr Leu Leu Glu Thr Asn Arg Phe Leu Asp 595 600 605 aac agcatc gac acc gcc tgg cta gat gcc ttg atc gca gag cgt gtg 1872 Asn Ser IleAsp Thr Ala Trp Leu Asp Ala Leu Ile Ala Glu Arg Val 610 615 620 caa tccgag aag ccg gat atc ctg ttg ggc gta atg tgc gga tcg ctg 1920 Gln Ser GluLys Pro Asp Ile Leu Leu Gly Val Met Cys Gly Ser Leu 625 630 635 640 cacatc gca gat cgt caa att act gag agc ttt tcc agc ttc caa acc 1968 His IleAla Asp Arg Gln Ile Thr Glu Ser Phe Ser Ser Phe Gln Thr 645 650 655 tctctg gag aaa ggt cag atc caa gca gcg aac acg ctg acg aac gtg 2016 Ser LeuGlu Lys Gly Gln Ile Gln Ala Ala Asn Thr Leu Thr Asn Val 660 665 670 gtggat gtt gag cta atc aac gat ggc atc cgt tac aag gtg cag gcc 2064 Val AspVal Glu Leu Ile Asn Asp Gly Ile Arg Tyr Lys Val Gln Ala 675 680 685 gccaag agc gga gcc aac tcg tac ttc ctg ctg atg aac agc tcg ttt 2112 Ala LysSer Gly Ala Asn Ser Tyr Phe Leu Leu Met Asn Ser Ser Phe 690 695 700 aaggag atc gag gtg cac cgc ctc tcc gac gga ggc ttg ctc atc tct 2160 Lys GluIle Glu Val His Arg Leu Ser Asp Gly Gly Leu Leu Ile Ser 705 710 715 720ttg gag ggc gcc tcc tac acc acg tac atg aag gag gag gtg gat cgc 2208 LeuGlu Gly Ala Ser Tyr Thr Thr Tyr Met Lys Glu Glu Val Asp Arg 725 730 735tac cgc att gtg att ggc aac cag aca tgt gtc ttt gaa aag gag aac 2256 TyrArg Ile Val Ile Gly Asn Gln Thr Cys Val Phe Glu Lys Glu Asn 740 745 750gat cca tcg ctg ttg cgc agt ccg tct gcg gga aag ctc atc aac atg 2304 AspPro Ser Leu Leu Arg Ser Pro Ser Ala Gly Lys Leu Ile Asn Met 755 760 765att gtg gaa gat ggc gct cat gta agc aag ggc cag gcc tat gct gag 2352 IleVal Glu Asp Gly Ala His Val Ser Lys Gly Gln Ala Tyr Ala Glu 770 775 780att gag gtg atg aag atg gtg atg acc ctg acg tcc cag gag gca ggc 2400 IleGlu Val Met Lys Met Val Met Thr Leu Thr Ser Gln Glu Ala Gly 785 790 795800 aca gtg aca ttt gtg cgt cga cca gga gct gtt cta gat gca gga tcc 2448Thr Val Thr Phe Val Arg Arg Pro Gly Ala Val Leu Asp Ala Gly Ser 805 810815 ctt ttg ggc cac ttg gag ctg gac gat cca tcg ctg gtg acg aaa gcg 2496Leu Leu Gly His Leu Glu Leu Asp Asp Pro Ser Leu Val Thr Lys Ala 820 825830 cag ccc ttc aag gga cag ttc ctg cag cca gag aac gca ccg gta ccc 2544Gln Pro Phe Lys Gly Gln Phe Leu Gln Pro Glu Asn Ala Pro Val Pro 835 840845 gag aaa cta aac agg gtg cac aat act tac aag agt atc ctt gaa aac 2592Glu Lys Leu Asn Arg Val His Asn Thr Tyr Lys Ser Ile Leu Glu Asn 850 855860 aca ctg gct ggt tac tgc ctg cca gaa ccg ttc aat gca cag cga ctc 2640Thr Leu Ala Gly Tyr Cys Leu Pro Glu Pro Phe Asn Ala Gln Arg Leu 865 870875 880 aga gac atc atc gaa aaa ttc atg caa agc ttg cgt gat ccc tcg ttg2688 Arg Asp Ile Ile Glu Lys Phe Met Gln Ser Leu Arg Asp Pro Ser Leu 885890 895 ccg ttg ttg gag ctg caa gaa gtt atc gcc tcc atc tct ggt cgc ata2736 Pro Leu Leu Glu Leu Gln Glu Val Ile Ala Ser Ile Ser Gly Arg Ile 900905 910 ccc ata tcc gtg gag aag aag atc cgg aaa ctg atg acg ctg tac gag2784 Pro Ile Ser Val Glu Lys Lys Ile Arg Lys Leu Met Thr Leu Tyr Glu 915920 925 cga aac ata act agt gtc ctg gcc caa ttc ccc tcg cag cag atc gcc2832 Arg Asn Ile Thr Ser Val Leu Ala Gln Phe Pro Ser Gln Gln Ile Ala 930935 940 agt gtt att gac agc cat gcg gcc acg ctg cag aag cgc gct gac cgt2880 Ser Val Ile Asp Ser His Ala Ala Thr Leu Gln Lys Arg Ala Asp Arg 945950 955 960 gat gtc ttc ttc ctg acc acc cag agc att gtg cag ctg gtg cagcgc 2928 Asp Val Phe Phe Leu Thr Thr Gln Ser Ile Val Gln Leu Val Gln Arg965 970 975 tat agg aac gga atc cgc ggc aga atg aag gcc gcc gtt cat gagctg 2976 Tyr Arg Asn Gly Ile Arg Gly Arg Met Lys Ala Ala Val His Glu Leu980 985 990 ttg cgt cag tac tac gat gta gag tcg cag ttc cag tat gga cactac 3024 Leu Arg Gln Tyr Tyr Asp Val Glu Ser Gln Phe Gln Tyr Gly His Tyr995 1000 1005 gac aaa tgc gtg gga ctg gtg cga gag cac aac aag gac gacatg 3069 Asp Lys Cys Val Gly Leu Val Arg Glu His Asn Lys Asp Asp Met1010 1015 1020 cag acg gtg gtc aac acc atc ttc tcg cac tct cag gtg gccaag 3114 Gln Thr Val Val Asn Thr Ile Phe Ser His Ser Gln Val Ala Lys1025 1030 1035 aag aat ctg ctg gtc act ctg ctc att gat cac ctg tgg gccaac 3159 Lys Asn Leu Leu Val Thr Leu Leu Ile Asp His Leu Trp Ala Asn1040 1045 1050 gaa cct gga cta acg gac gaa ttg gcc aac acg cta agt gaattg 3204 Glu Pro Gly Leu Thr Asp Glu Leu Ala Asn Thr Leu Ser Glu Leu1055 1060 1065 acc tct ttg aat cga gct gag cac tct agg gtt gcc ctg cggtcc 3249 Thr Ser Leu Asn Arg Ala Glu His Ser Arg Val Ala Leu Arg Ser1070 1075 1080 cgc caa gtt ctg atc gct gcc cac cag ccg gct tat gag ctgcgc 3294 Arg Gln Val Leu Ile Ala Ala His Gln Pro Ala Tyr Glu Leu Arg1085 1090 1095 cac aac caa atg gag tcg atc ttt ctc tcc gcc gtt gac atgtac 3339 His Asn Gln Met Glu Ser Ile Phe Leu Ser Ala Val Asp Met Tyr1100 1105 1110 ggt cat gac ttc cac ccc gag aac ctg cag cgc ctg att ctgtcg 3384 Gly His Asp Phe His Pro Glu Asn Leu Gln Arg Leu Ile Leu Ser1115 1120 1125 gag acc tca atc ttt gac atc ctg cac gac ttc ttc tac cactct 3429 Glu Thr Ser Ile Phe Asp Ile Leu His Asp Phe Phe Tyr His Ser1130 1135 1140 aac cgg gca gtg tgc aat gct gct ctg gaa gtc tat gtg aggaga 3474 Asn Arg Ala Val Cys Asn Ala Ala Leu Glu Val Tyr Val Arg Arg1145 1150 1155 gct tac aca tcc tat gag ctg acc tgc ttg cag cat ttg gaactc 3519 Ala Tyr Thr Ser Tyr Glu Leu Thr Cys Leu Gln His Leu Glu Leu1160 1165 1170 tcc ggt ggc ctg ccg ctg gtg cac ttc cag ttc ctc ctc cccaca 3564 Ser Gly Gly Leu Pro Leu Val His Phe Gln Phe Leu Leu Pro Thr1175 1180 1185 gct cac ccg aac aga ctg ttc tcg cgc atg tcc tcc ccc gatgga 3609 Ala His Pro Asn Arg Leu Phe Ser Arg Met Ser Ser Pro Asp Gly1190 1195 1200 ttg gat cag gca gcg gca gag tct ttg gga aac tca ttc gtgcgc 3654 Leu Asp Gln Ala Ala Ala Glu Ser Leu Gly Asn Ser Phe Val Arg1205 1210 1215 acc gga gcg att gca gcc ttt gac tcc ttc gaa cac ttt gagatg 3699 Thr Gly Ala Ile Ala Ala Phe Asp Ser Phe Glu His Phe Glu Met1220 1225 1230 tac tcg gac gag att ctg gat ctg ctc gaa gac ttc gtc tcgcca 3744 Tyr Ser Asp Glu Ile Leu Asp Leu Leu Glu Asp Phe Val Ser Pro1235 1240 1245 gcc atg gtt aat gcc aag gtc ctg gaa gcc gta gag gca gcggat 3789 Ala Met Val Asn Ala Lys Val Leu Glu Ala Val Glu Ala Ala Asp1250 1255 1260 tct atc tcg gac agc cga cac agc acc tcg atc aat gtg tcgttg 3834 Ser Ile Ser Asp Ser Arg His Ser Thr Ser Ile Asn Val Ser Leu1265 1270 1275 tcg gat ccc gta acc cgg gcg aat gct gcc gag gag gcc aagtcc 3879 Ser Asp Pro Val Thr Arg Ala Asn Ala Ala Glu Glu Ala Lys Ser1280 1285 1290 acc gaa ccg att cac att gtt agt gtg gct gtg aga gaa acgggg 3924 Thr Glu Pro Ile His Ile Val Ser Val Ala Val Arg Glu Thr Gly1295 1300 1305 gag ttg gat gac ctg caa atg gcc caa atc ttt gga aat tattgc 3969 Glu Leu Asp Asp Leu Gln Met Ala Gln Ile Phe Gly Asn Tyr Cys1310 1315 1320 caa gag cat aac gag gag ctc ttc cag cga cgc att cgt aggatt 4014 Gln Glu His Asn Glu Glu Leu Phe Gln Arg Arg Ile Arg Arg Ile1325 1330 1335 aca ttt gct gct ctg aag aag cgg caa ttc ccc aag ttc tttacg 4059 Thr Phe Ala Ala Leu Lys Lys Arg Gln Phe Pro Lys Phe Phe Thr1340 1345 1350 ttc aga gcc aga gat aag ttc acg gag gat cgt att tac cggcat 4104 Phe Arg Ala Arg Asp Lys Phe Thr Glu Asp Arg Ile Tyr Arg His1355 1360 1365 ctg gag cca gca tct gct ttc cat ctg gag ctg aac cgc atgaag 4149 Leu Glu Pro Ala Ser Ala Phe His Leu Glu Leu Asn Arg Met Lys1370 1375 1380 acg tac gat ctg gag gct ctg ccc acg gct aac caa aag atgcac 4194 Thr Tyr Asp Leu Glu Ala Leu Pro Thr Ala Asn Gln Lys Met His1385 1390 1395 ctg tac ctt ggc aag gcc aag gtt tcg aaa ggt caa gag gtcacg 4239 Leu Tyr Leu Gly Lys Ala Lys Val Ser Lys Gly Gln Glu Val Thr1400 1405 1410 gac tac cgc ttc ttc att cgc tcg atc atc cgt cat tcg gatctg 4284 Asp Tyr Arg Phe Phe Ile Arg Ser Ile Ile Arg His Ser Asp Leu1415 1420 1425 att acc aag gaa gcc tct ttc gag tat ctg caa aac gaa ggagag 4329 Ile Thr Lys Glu Ala Ser Phe Glu Tyr Leu Gln Asn Glu Gly Glu1430 1435 1440 cgt gtg ctc ctg gag gcc atg gat gag ctg gag gtg gca ttctcg 4374 Arg Val Leu Leu Glu Ala Met Asp Glu Leu Glu Val Ala Phe Ser1445 1450 1455 cat ccg cac gcc aaa cgc acg gac tgc aac cac atc ttc ctgaac 4419 His Pro His Ala Lys Arg Thr Asp Cys Asn His Ile Phe Leu Asn1460 1465 1470 ttt gtg ccc acc gtc atc atg gat ccg gct aag atc gag gaatct 4464 Phe Val Pro Thr Val Ile Met Asp Pro Ala Lys Ile Glu Glu Ser1475 1480 1485 gta aca aag atg att atg cga tat ggt cca cgt ctt tgg aagctg 4509 Val Thr Lys Met Ile Met Arg Tyr Gly Pro Arg Leu Trp Lys Leu1490 1495 1500 cgt gta ctg cag gct gag ctc aag atg gtc atc cgc cag tcacca 4554 Arg Val Leu Gln Ala Glu Leu Lys Met Val Ile Arg Gln Ser Pro1505 1510 1515 cag tca ccc act cag gca gtg cgt ctg tgc att gca aat gactcc 4599 Gln Ser Pro Thr Gln Ala Val Arg Leu Cys Ile Ala Asn Asp Ser1520 1525 1530 ggc tac ttc ctg gat att tcg atg tat acc gaa caa aca gaacca 4644 Gly Tyr Phe Leu Asp Ile Ser Met Tyr Thr Glu Gln Thr Glu Pro1535 1540 1545 gag aca gga atc att aag ttt aag gcc tac ggt gag aag caggga 4689 Glu Thr Gly Ile Ile Lys Phe Lys Ala Tyr Gly Glu Lys Gln Gly1550 1555 1560 tct ctg cac gga cat ccc att tcg acg ccc tac atg acc aaggac 4734 Ser Leu His Gly His Pro Ile Ser Thr Pro Tyr Met Thr Lys Asp1565 1570 1575 ttc ctg cag cag aaa cgt ttc cag gcg cag tcc aat ggt accacc 4779 Phe Leu Gln Gln Lys Arg Phe Gln Ala Gln Ser Asn Gly Thr Thr1580 1585 1590 tat gtc tat gat gtg ccc gac atg ttc cgc cag atg acc gagcgt 4824 Tyr Val Tyr Asp Val Pro Asp Met Phe Arg Gln Met Thr Glu Arg1595 1600 1605 cac tgg aga gaa ttc tcc aag gct cgt ccc acc gtg gac attcgc 4869 His Trp Arg Glu Phe Ser Lys Ala Arg Pro Thr Val Asp Ile Arg1610 1615 1620 act ccc gac aag att ttg atc gag tgc aag gag ctg gtc ctcgag 4914 Thr Pro Asp Lys Ile Leu Ile Glu Cys Lys Glu Leu Val Leu Glu1625 1630 1635 ggc gac aac ctt gta gag atg cag cgt ctg ccc ggc gaa aacaat 4959 Gly Asp Asn Leu Val Glu Met Gln Arg Leu Pro Gly Glu Asn Asn1640 1645 1650 tgc ggc atg gtg gct tgg cgc att gtc ttg gct act ccg gaatat 5004 Cys Gly Met Val Ala Trp Arg Ile Val Leu Ala Thr Pro Glu Tyr1655 1660 1665 ccg aat ggc cgc gag atc att gtt ata gcc aac gat ctc acctac 5049 Pro Asn Gly Arg Glu Ile Ile Val Ile Ala Asn Asp Leu Thr Tyr1670 1675 1680 ttg att ggt tcc ttt gga att aag gag gac gtt ctc ttt gccaag 5094 Leu Ile Gly Ser Phe Gly Ile Lys Glu Asp Val Leu Phe Ala Lys1685 1690 1695 gct tcc caa ttg gct cgc caa ctc aaa gta ccg agg ata tacatc 5139 Ala Ser Gln Leu Ala Arg Gln Leu Lys Val Pro Arg Ile Tyr Ile1700 1705 1710 tcc gtt aac agc ggt gcc cgc ata gga ctt gct gag gag gttaaa 5184 Ser Val Asn Ser Gly Ala Arg Ile Gly Leu Ala Glu Glu Val Lys1715 1720 1725 gct atg ttc aag atc gca tgg gag gat cca gag gag cca gataag 5229 Ala Met Phe Lys Ile Ala Trp Glu Asp Pro Glu Glu Pro Asp Lys1730 1735 1740 ggc ttc aag tac ctc tac ttg agc acc gag gac tac gcc caggtg 5274 Gly Phe Lys Tyr Leu Tyr Leu Ser Thr Glu Asp Tyr Ala Gln Val1745 1750 1755 gcc aac ctg aac tcg gtg agg gct atc ctg atc gag gac gagggc 5319 Ala Asn Leu Asn Ser Val Arg Ala Ile Leu Ile Glu Asp Glu Gly1760 1765 1770 gag cag cgt tac aag att acc gac atc atc ggc aag gac gatggt 5364 Glu Gln Arg Tyr Lys Ile Thr Asp Ile Ile Gly Lys Asp Asp Gly1775 1780 1785 ctg ggc gtg gag aat ctg cgt tac gcc ggc ttg att gcc ggtgaa 5409 Leu Gly Val Glu Asn Leu Arg Tyr Ala Gly Leu Ile Ala Gly Glu1790 1795 1800 acg tcg cag gcc tac gag gag att gtt act atc gct atg gttacc 5454 Thr Ser Gln Ala Tyr Glu Glu Ile Val Thr Ile Ala Met Val Thr1805 1810 1815 tgc cgt acc att ggc att gga tcc tat gtg gtg cgc ctg ggtcag 5499 Cys Arg Thr Ile Gly Ile Gly Ser Tyr Val Val Arg Leu Gly Gln1820 1825 1830 cgc gtt atc cag atc gat aat tca cac att ata ctc act ggctat 5544 Arg Val Ile Gln Ile Asp Asn Ser His Ile Ile Leu Thr Gly Tyr1835 1840 1845 gct gcg ctt aac aag ctg ctt gga cgc aag gtg tat gcc tctaat 5589 Ala Ala Leu Asn Lys Leu Leu Gly Arg Lys Val Tyr Ala Ser Asn1850 1855 1860 aat cag ttg ggt ggc aca cag atc atg ttt aac aac gga gtcacc 5634 Asn Gln Leu Gly Gly Thr Gln Ile Met Phe Asn Asn Gly Val Thr1865 1870 1875 cac aaa aca gag gcc atc gac ttg gac ggt gtc tac acc atcctc 5679 His Lys Thr Glu Ala Ile Asp Leu Asp Gly Val Tyr Thr Ile Leu1880 1885 1890 gac tgg ctc tcg tac atc ccc gcg tac atc ggt tgt gac ctgccc 5724 Asp Trp Leu Ser Tyr Ile Pro Ala Tyr Ile Gly Cys Asp Leu Pro1895 1900 1905 att gtt ttg ccc aac gat cgt atc gaa cgc cct gtc gac ttcatg 5769 Ile Val Leu Pro Asn Asp Arg Ile Glu Arg Pro Val Asp Phe Met1910 1915 1920 ccc acc aag tcg ccc tac gat ccg cgc tgg atg ctg ggt ggccgt 5814 Pro Thr Lys Ser Pro Tyr Asp Pro Arg Trp Met Leu Gly Gly Arg1925 1930 1935 gtg aat ccc gtg aac gct aat gac tgg gag aac gga ttc tttgat 5859 Val Asn Pro Val Asn Ala Asn Asp Trp Glu Asn Gly Phe Phe Asp1940 1945 1950 cgc gac tcg tgg agc gaa atc atg gcc tcg tgg gcc aag acagtg 5904 Arg Asp Ser Trp Ser Glu Ile Met Ala Ser Trp Ala Lys Thr Val1955 1960 1965 gtc act ggt cgc gca cgt cta ggc ggt gtc ccc gtg ggc gtaata 5949 Val Thr Gly Arg Ala Arg Leu Gly Gly Val Pro Val Gly Val Ile1970 1975 1980 gcc gtt gag acc cgc acc gta gaa gtg gag atg ccc gcc gatcct 5994 Ala Val Glu Thr Arg Thr Val Glu Val Glu Met Pro Ala Asp Pro1985 1990 1995 gcc aat ctc gat tcg gaa gcc aag acc ctg cag cag gca ggtcag 6039 Ala Asn Leu Asp Ser Glu Ala Lys Thr Leu Gln Gln Ala Gly Gln2000 2005 2010 gtg tgg tac ccc gac tcc tcg tac aaa acg gca caa gcg atcaaa 6084 Val Trp Tyr Pro Asp Ser Ser Tyr Lys Thr Ala Gln Ala Ile Lys2015 2020 2025 gat ttt gga cga gag gag ttg ccg ctg att gtt ttc gca aattgg 6129 Asp Phe Gly Arg Glu Glu Leu Pro Leu Ile Val Phe Ala Asn Trp2030 2035 2040 cga ggc ttc tcc ggt ggc atg aag gac atg tac gag caa atcgtc 6174 Arg Gly Phe Ser Gly Gly Met Lys Asp Met Tyr Glu Gln Ile Val2045 2050 2055 aag ttc gga gca tac att gtc gac ggc ctg cgg gag tac aagaag 6219 Lys Phe Gly Ala Tyr Ile Val Asp Gly Leu Arg Glu Tyr Lys Lys2060 2065 2070 cct gtg ctc atc tac ctg ccg ccc aac gcc gag ctg cga ggtgga 6264 Pro Val Leu Ile Tyr Leu Pro Pro Asn Ala Glu Leu Arg Gly Gly2075 2080 2085 gcc tgg gcc gtg ttg gat tcc ctc att aac ccg cgc tac atggaa 6309 Ala Trp Ala Val Leu Asp Ser Leu Ile Asn Pro Arg Tyr Met Glu2090 2095 2100 acg tat gcc gat ccg gag gcc aga gga gga gtt ctc gag ccggag 6354 Thr Tyr Ala Asp Pro Glu Ala Arg Gly Gly Val Leu Glu Pro Glu2105 2110 2115 ggc att gtg gaa ata aag tac aaa gag aag gac ctg gtc aagacg 6399 Gly Ile Val Glu Ile Lys Tyr Lys Glu Lys Asp Leu Val Lys Thr2120 2125 2130 att cac cgc ttg gat ccg acc acc att gcg ctg aaa aag gagctc 6444 Ile His Arg Leu Asp Pro Thr Thr Ile Ala Leu Lys Lys Glu Leu2135 2140 2145 gat gag gca aat gcg tct ggc gac aag gtc agg gct gct caggtg 6489 Asp Glu Ala Asn Ala Ser Gly Asp Lys Val Arg Ala Ala Gln Val2150 2155 2160 gac gaa aag atc aag gcc cgc atc gct gtg cta atg cac gtctac 6534 Asp Glu Lys Ile Lys Ala Arg Ile Ala Val Leu Met His Val Tyr2165 2170 2175 cac acg gta gca gtt cac ttt gcc gac ctg cac gac acg ccggag 6579 His Thr Val Ala Val His Phe Ala Asp Leu His Asp Thr Pro Glu2180 2185 2190 cga atg cta gag aag gag tgt atc agt gag att gtg cct tggcgc 6624 Arg Met Leu Glu Lys Glu Cys Ile Ser Glu Ile Val Pro Trp Arg2195 2200 2205 gat tcc cgc cgc tgg ctg tac tgg cgt ctg cga cgt ctc ctgttg 6669 Asp Ser Arg Arg Trp Leu Tyr Trp Arg Leu Arg Arg Leu Leu Leu2210 2215 2220 gag gac gca tat att aag aag atc ctg cgc gct cag gac aacctc 6714 Glu Asp Ala Tyr Ile Lys Lys Ile Leu Arg Ala Gln Asp Asn Leu2225 2230 2235 tcc gtg ggt cag gcc aag cag atg ctg cgt cga tgg ctg gtagag 6759 Ser Val Gly Gln Ala Lys Gln Met Leu Arg Arg Trp Leu Val Glu2240 2245 2250 gag aag ggt gcc aca gag gct tat ctg tgg gac aaa aac gaggag 6804 Glu Lys Gly Ala Thr Glu Ala Tyr Leu Trp Asp Lys Asn Glu Glu2255 2260 2265 atg gtg tct tgg tat gag gag cag atc aat gcc gaa tct attgtt 6849 Met Val Ser Trp Tyr Glu Glu Gln Ile Asn Ala Glu Ser Ile Val2270 2275 2280 tcc cgc aac gtg aac tcc gtg aga cgg gat gcc att att tctacc 6894 Ser Arg Asn Val Asn Ser Val Arg Arg Asp Ala Ile Ile Ser Thr2285 2290 2295 att tcg aaa atg ctc gag gac tgt ccc gac gta gcg ctg gacgct 6939 Ile Ser Lys Met Leu Glu Asp Cys Pro Asp Val Ala Leu Asp Ala2300 2305 2310 gtt gtg ggt ctt tgc caa ggt ctg acg cca gtg aat cga ggcgtg 6984 Val Val Gly Leu Cys Gln Gly Leu Thr Pro Val Asn Arg Gly Val2315 2320 2325 gtc gta cgc aca tta gcc cag atg cag ctg aat gag gag acctct 7029 Val Val Arg Thr Leu Ala Gln Met Gln Leu Asn Glu Glu Thr Ser2330 2335 2340 aac agc aac cag gga tga 7047 Asn Ser Asn Gln Gly 2345 22348 PRT Drosophila melanogaster 2 Met Leu Lys Arg Arg Ala Ser Lys ArgPhe Val Leu Val Glu Ser Gly 1 5 10 15 Glu Asp Asn Ala Asn Gly Ser GlySer Ala Ser Gly Ser Gly Ser Gly 20 25 30 Ser Gly Val Gly Thr Ala Val IlePro Gln Phe Val Ala Val Asp Cys 35 40 45 Gly Gln Asn Glu Ser Asn Asn AsnHis Val Gly Glu Met Ser Ala Ser 50 55 60 Ile Ser Asn His Asn Ser Ser AsnAsn Gln Ser Ser Pro Ser Leu Leu 65 70 75 80 Ser Val Pro Val Val Gly ThrLeu Lys Pro Ser Met Ser Arg Gly Thr 85 90 95 Gly Leu Gly Gln Asp Arg HisGln Asp Arg Asp Phe His Ile Ala Thr 100 105 110 Thr Glu Glu Phe Val LysArg Phe Gly Gly Thr Arg Val Ile Asn Lys 115 120 125 Val Leu Ile Ala AsnAsn Gly Ile Ala Ala Val Lys Cys Met Arg Ser 130 135 140 Ile Arg Arg TrpAla Tyr Glu Met Phe Lys Asn Glu Arg Ala Ile Arg 145 150 155 160 Phe ValVal Met Val Thr Pro Glu Asp Leu Lys Ala Asn Ala Glu Tyr 165 170 175 IleLys Met Ala Asp His Tyr Val Pro Val Pro Gly Gly Ser Asn Asn 180 185 190Asn Asn Tyr Ala Asn Val Glu Leu Ile Val Asp Ile Ala Leu Arg Thr 195 200205 Gln Val Gln Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro 210215 220 Lys Leu Pro Glu Leu Leu His Lys Glu Gly Leu Val Phe Leu Gly Pro225 230 235 240 Pro Glu Arg Ala Met Trp Ala Leu Gly Asp Lys Val Ala SerSer Ile 245 250 255 Val Ala Gln Thr Ala Glu Ile Pro Thr Leu Pro Trp SerGly Ser Asp 260 265 270 Leu Lys Ala Gln Tyr Ser Gly Lys Lys Ile Lys IleSer Ser Glu Leu 275 280 285 Phe Ala Arg Gly Cys Val Thr Asn Val Glu GlnGly Leu Ala Ala Val 290 295 300 Asn Lys Ile Gly Phe Pro Val Met Ile LysAla Ser Glu Gly Gly Gly 305 310 315 320 Gly Lys Gly Ile Arg Arg Val AspThr Thr Glu Glu Phe Pro Gly Leu 325 330 335 Phe Arg Gln Val Gln Ala GluVal Pro Gly Ser Pro Ile Phe Val Met 340 345 350 Lys Leu Ala Arg Gly AlaArg His Leu Glu Val Gln Leu Leu Ala Asp 355 360 365 Gln Tyr Gly Asn AlaIle Ser Leu Phe Gly Arg Asp Cys Ser Ile Gln 370 375 380 Arg Arg His GlnLys Ile Ile Glu Glu Ala Pro Ala Ile Val Ala Gln 385 390 395 400 Pro GluVal Phe Glu Asp Met Glu Lys Ala Ala Val Arg Leu Ala Lys 405 410 415 MetVal Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr Asp Pro 420 425 430Glu Gly Arg Tyr Phe Phe Leu Glu Leu Asn Pro Arg Leu Gln Val Glu 435 440445 His Pro Cys Thr Glu Met Val Ala Asp Val Asn Leu Pro Ala Ala Gln 450455 460 Leu Gln Ile Gly Met Gly Ile Pro Leu Tyr Arg Leu Lys Asp Ile Arg465 470 475 480 Leu Leu Tyr Gly Glu Ser Pro Trp Gly Ser Ser Val Ile AspPhe Glu 485 490 495 Asn Pro Pro Asn Lys Pro Arg Pro Ser Gly His Val IleAla Ala Arg 500 505 510 Ile Thr Ser Glu Asn Pro Asp Glu Gly Phe Lys ProSer Ser Gly Thr 515 520 525 Val Gln Glu Leu Asn Phe Arg Ser Ser Lys AsnVal Trp Gly Tyr Phe 530 535 540 Ser Val Ala Ala Ser Gly Gly Leu His GluPhe Ala Asp Ser Gln Phe 545 550 555 560 Gly His Cys Phe Ser Trp Gly GluAsn Arg Gln Gln Ala Arg Glu Asn 565 570 575 Leu Val Ile Ala Leu Lys GluLeu Ser Ile Arg Gly Asp Phe Arg Thr 580 585 590 Thr Val Glu Tyr Leu IleThr Leu Leu Glu Thr Asn Arg Phe Leu Asp 595 600 605 Asn Ser Ile Asp ThrAla Trp Leu Asp Ala Leu Ile Ala Glu Arg Val 610 615 620 Gln Ser Glu LysPro Asp Ile Leu Leu Gly Val Met Cys Gly Ser Leu 625 630 635 640 His IleAla Asp Arg Gln Ile Thr Glu Ser Phe Ser Ser Phe Gln Thr 645 650 655 SerLeu Glu Lys Gly Gln Ile Gln Ala Ala Asn Thr Leu Thr Asn Val 660 665 670Val Asp Val Glu Leu Ile Asn Asp Gly Ile Arg Tyr Lys Val Gln Ala 675 680685 Ala Lys Ser Gly Ala Asn Ser Tyr Phe Leu Leu Met Asn Ser Ser Phe 690695 700 Lys Glu Ile Glu Val His Arg Leu Ser Asp Gly Gly Leu Leu Ile Ser705 710 715 720 Leu Glu Gly Ala Ser Tyr Thr Thr Tyr Met Lys Glu Glu ValAsp Arg 725 730 735 Tyr Arg Ile Val Ile Gly Asn Gln Thr Cys Val Phe GluLys Glu Asn 740 745 750 Asp Pro Ser Leu Leu Arg Ser Pro Ser Ala Gly LysLeu Ile Asn Met 755 760 765 Ile Val Glu Asp Gly Ala His Val Ser Lys GlyGln Ala Tyr Ala Glu 770 775 780 Ile Glu Val Met Lys Met Val Met Thr LeuThr Ser Gln Glu Ala Gly 785 790 795 800 Thr Val Thr Phe Val Arg Arg ProGly Ala Val Leu Asp Ala Gly Ser 805 810 815 Leu Leu Gly His Leu Glu LeuAsp Asp Pro Ser Leu Val Thr Lys Ala 820 825 830 Gln Pro Phe Lys Gly GlnPhe Leu Gln Pro Glu Asn Ala Pro Val Pro 835 840 845 Glu Lys Leu Asn ArgVal His Asn Thr Tyr Lys Ser Ile Leu Glu Asn 850 855 860 Thr Leu Ala GlyTyr Cys Leu Pro Glu Pro Phe Asn Ala Gln Arg Leu 865 870 875 880 Arg AspIle Ile Glu Lys Phe Met Gln Ser Leu Arg Asp Pro Ser Leu 885 890 895 ProLeu Leu Glu Leu Gln Glu Val Ile Ala Ser Ile Ser Gly Arg Ile 900 905 910Pro Ile Ser Val Glu Lys Lys Ile Arg Lys Leu Met Thr Leu Tyr Glu 915 920925 Arg Asn Ile Thr Ser Val Leu Ala Gln Phe Pro Ser Gln Gln Ile Ala 930935 940 Ser Val Ile Asp Ser His Ala Ala Thr Leu Gln Lys Arg Ala Asp Arg945 950 955 960 Asp Val Phe Phe Leu Thr Thr Gln Ser Ile Val Gln Leu ValGln Arg 965 970 975 Tyr Arg Asn Gly Ile Arg Gly Arg Met Lys Ala Ala ValHis Glu Leu 980 985 990 Leu Arg Gln Tyr Tyr Asp Val Glu Ser Gln Phe GlnTyr Gly His Tyr 995 1000 1005 Asp Lys Cys Val Gly Leu Val Arg Glu HisAsn Lys Asp Asp Met 1010 1015 1020 Gln Thr Val Val Asn Thr Ile Phe SerHis Ser Gln Val Ala Lys 1025 1030 1035 Lys Asn Leu Leu Val Thr Leu LeuIle Asp His Leu Trp Ala Asn 1040 1045 1050 Glu Pro Gly Leu Thr Asp GluLeu Ala Asn Thr Leu Ser Glu Leu 1055 1060 1065 Thr Ser Leu Asn Arg AlaGlu His Ser Arg Val Ala Leu Arg Ser 1070 1075 1080 Arg Gln Val Leu IleAla Ala His Gln Pro Ala Tyr Glu Leu Arg 1085 1090 1095 His Asn Gln MetGlu Ser Ile Phe Leu Ser Ala Val Asp Met Tyr 1100 1105 1110 Gly His AspPhe His Pro Glu Asn Leu Gln Arg Leu Ile Leu Ser 1115 1120 1125 Glu ThrSer Ile Phe Asp Ile Leu His Asp Phe Phe Tyr His Ser 1130 1135 1140 AsnArg Ala Val Cys Asn Ala Ala Leu Glu Val Tyr Val Arg Arg 1145 1150 1155Ala Tyr Thr Ser Tyr Glu Leu Thr Cys Leu Gln His Leu Glu Leu 1160 11651170 Ser Gly Gly Leu Pro Leu Val His Phe Gln Phe Leu Leu Pro Thr 11751180 1185 Ala His Pro Asn Arg Leu Phe Ser Arg Met Ser Ser Pro Asp Gly1190 1195 1200 Leu Asp Gln Ala Ala Ala Glu Ser Leu Gly Asn Ser Phe ValArg 1205 1210 1215 Thr Gly Ala Ile Ala Ala Phe Asp Ser Phe Glu His PheGlu Met 1220 1225 1230 Tyr Ser Asp Glu Ile Leu Asp Leu Leu Glu Asp PheVal Ser Pro 1235 1240 1245 Ala Met Val Asn Ala Lys Val Leu Glu Ala ValGlu Ala Ala Asp 1250 1255 1260 Ser Ile Ser Asp Ser Arg His Ser Thr SerIle Asn Val Ser Leu 1265 1270 1275 Ser Asp Pro Val Thr Arg Ala Asn AlaAla Glu Glu Ala Lys Ser 1280 1285 1290 Thr Glu Pro Ile His Ile Val SerVal Ala Val Arg Glu Thr Gly 1295 1300 1305 Glu Leu Asp Asp Leu Gln MetAla Gln Ile Phe Gly Asn Tyr Cys 1310 1315 1320 Gln Glu His Asn Glu GluLeu Phe Gln Arg Arg Ile Arg Arg Ile 1325 1330 1335 Thr Phe Ala Ala LeuLys Lys Arg Gln Phe Pro Lys Phe Phe Thr 1340 1345 1350 Phe Arg Ala ArgAsp Lys Phe Thr Glu Asp Arg Ile Tyr Arg His 1355 1360 1365 Leu Glu ProAla Ser Ala Phe His Leu Glu Leu Asn Arg Met Lys 1370 1375 1380 Thr TyrAsp Leu Glu Ala Leu Pro Thr Ala Asn Gln Lys Met His 1385 1390 1395 LeuTyr Leu Gly Lys Ala Lys Val Ser Lys Gly Gln Glu Val Thr 1400 1405 1410Asp Tyr Arg Phe Phe Ile Arg Ser Ile Ile Arg His Ser Asp Leu 1415 14201425 Ile Thr Lys Glu Ala Ser Phe Glu Tyr Leu Gln Asn Glu Gly Glu 14301435 1440 Arg Val Leu Leu Glu Ala Met Asp Glu Leu Glu Val Ala Phe Ser1445 1450 1455 His Pro His Ala Lys Arg Thr Asp Cys Asn His Ile Phe LeuAsn 1460 1465 1470 Phe Val Pro Thr Val Ile Met Asp Pro Ala Lys Ile GluGlu Ser 1475 1480 1485 Val Thr Lys Met Ile Met Arg Tyr Gly Pro Arg LeuTrp Lys Leu 1490 1495 1500 Arg Val Leu Gln Ala Glu Leu Lys Met Val IleArg Gln Ser Pro 1505 1510 1515 Gln Ser Pro Thr Gln Ala Val Arg Leu CysIle Ala Asn Asp Ser 1520 1525 1530 Gly Tyr Phe Leu Asp Ile Ser Met TyrThr Glu Gln Thr Glu Pro 1535 1540 1545 Glu Thr Gly Ile Ile Lys Phe LysAla Tyr Gly Glu Lys Gln Gly 1550 1555 1560 Ser Leu His Gly His Pro IleSer Thr Pro Tyr Met Thr Lys Asp 1565 1570 1575 Phe Leu Gln Gln Lys ArgPhe Gln Ala Gln Ser Asn Gly Thr Thr 1580 1585 1590 Tyr Val Tyr Asp ValPro Asp Met Phe Arg Gln Met Thr Glu Arg 1595 1600 1605 His Trp Arg GluPhe Ser Lys Ala Arg Pro Thr Val Asp Ile Arg 1610 1615 1620 Thr Pro AspLys Ile Leu Ile Glu Cys Lys Glu Leu Val Leu Glu 1625 1630 1635 Gly AspAsn Leu Val Glu Met Gln Arg Leu Pro Gly Glu Asn Asn 1640 1645 1650 CysGly Met Val Ala Trp Arg Ile Val Leu Ala Thr Pro Glu Tyr 1655 1660 1665Pro Asn Gly Arg Glu Ile Ile Val Ile Ala Asn Asp Leu Thr Tyr 1670 16751680 Leu Ile Gly Ser Phe Gly Ile Lys Glu Asp Val Leu Phe Ala Lys 16851690 1695 Ala Ser Gln Leu Ala Arg Gln Leu Lys Val Pro Arg Ile Tyr Ile1700 1705 1710 Ser Val Asn Ser Gly Ala Arg Ile Gly Leu Ala Glu Glu ValLys 1715 1720 1725 Ala Met Phe Lys Ile Ala Trp Glu Asp Pro Glu Glu ProAsp Lys 1730 1735 1740 Gly Phe Lys Tyr Leu Tyr Leu Ser Thr Glu Asp TyrAla Gln Val 1745 1750 1755 Ala Asn Leu Asn Ser Val Arg Ala Ile Leu IleGlu Asp Glu Gly 1760 1765 1770 Glu Gln Arg Tyr Lys Ile Thr Asp Ile IleGly Lys Asp Asp Gly 1775 1780 1785 Leu Gly Val Glu Asn Leu Arg Tyr AlaGly Leu Ile Ala Gly Glu 1790 1795 1800 Thr Ser Gln Ala Tyr Glu Glu IleVal Thr Ile Ala Met Val Thr 1805 1810 1815 Cys Arg Thr Ile Gly Ile GlySer Tyr Val Val Arg Leu Gly Gln 1820 1825 1830 Arg Val Ile Gln Ile AspAsn Ser His Ile Ile Leu Thr Gly Tyr 1835 1840 1845 Ala Ala Leu Asn LysLeu Leu Gly Arg Lys Val Tyr Ala Ser Asn 1850 1855 1860 Asn Gln Leu GlyGly Thr Gln Ile Met Phe Asn Asn Gly Val Thr 1865 1870 1875 His Lys ThrGlu Ala Ile Asp Leu Asp Gly Val Tyr Thr Ile Leu 1880 1885 1890 Asp TrpLeu Ser Tyr Ile Pro Ala Tyr Ile Gly Cys Asp Leu Pro 1895 1900 1905 IleVal Leu Pro Asn Asp Arg Ile Glu Arg Pro Val Asp Phe Met 1910 1915 1920Pro Thr Lys Ser Pro Tyr Asp Pro Arg Trp Met Leu Gly Gly Arg 1925 19301935 Val Asn Pro Val Asn Ala Asn Asp Trp Glu Asn Gly Phe Phe Asp 19401945 1950 Arg Asp Ser Trp Ser Glu Ile Met Ala Ser Trp Ala Lys Thr Val1955 1960 1965 Val Thr Gly Arg Ala Arg Leu Gly Gly Val Pro Val Gly ValIle 1970 1975 1980 Ala Val Glu Thr Arg Thr Val Glu Val Glu Met Pro AlaAsp Pro 1985 1990 1995 Ala Asn Leu Asp Ser Glu Ala Lys Thr Leu Gln GlnAla Gly Gln 2000 2005 2010 Val Trp Tyr Pro Asp Ser Ser Tyr Lys Thr AlaGln Ala Ile Lys 2015 2020 2025 Asp Phe Gly Arg Glu Glu Leu Pro Leu IleVal Phe Ala Asn Trp 2030 2035 2040 Arg Gly Phe Ser Gly Gly Met Lys AspMet Tyr Glu Gln Ile Val 2045 2050 2055 Lys Phe Gly Ala Tyr Ile Val AspGly Leu Arg Glu Tyr Lys Lys 2060 2065 2070 Pro Val Leu Ile Tyr Leu ProPro Asn Ala Glu Leu Arg Gly Gly 2075 2080 2085 Ala Trp Ala Val Leu AspSer Leu Ile Asn Pro Arg Tyr Met Glu 2090 2095 2100 Thr Tyr Ala Asp ProGlu Ala Arg Gly Gly Val Leu Glu Pro Glu 2105 2110 2115 Gly Ile Val GluIle Lys Tyr Lys Glu Lys Asp Leu Val Lys Thr 2120 2125 2130 Ile His ArgLeu Asp Pro Thr Thr Ile Ala Leu Lys Lys Glu Leu 2135 2140 2145 Asp GluAla Asn Ala Ser Gly Asp Lys Val Arg Ala Ala Gln Val 2150 2155 2160 AspGlu Lys Ile Lys Ala Arg Ile Ala Val Leu Met His Val Tyr 2165 2170 2175His Thr Val Ala Val His Phe Ala Asp Leu His Asp Thr Pro Glu 2180 21852190 Arg Met Leu Glu Lys Glu Cys Ile Ser Glu Ile Val Pro Trp Arg 21952200 2205 Asp Ser Arg Arg Trp Leu Tyr Trp Arg Leu Arg Arg Leu Leu Leu2210 2215 2220 Glu Asp Ala Tyr Ile Lys Lys Ile Leu Arg Ala Gln Asp AsnLeu 2225 2230 2235 Ser Val Gly Gln Ala Lys Gln Met Leu Arg Arg Trp LeuVal Glu 2240 2245 2250 Glu Lys Gly Ala Thr Glu Ala Tyr Leu Trp Asp LysAsn Glu Glu 2255 2260 2265 Met Val Ser Trp Tyr Glu Glu Gln Ile Asn AlaGlu Ser Ile Val 2270 2275 2280 Ser Arg Asn Val Asn Ser Val Arg Arg AspAla Ile Ile Ser Thr 2285 2290 2295 Ile Ser Lys Met Leu Glu Asp Cys ProAsp Val Ala Leu Asp Ala 2300 2305 2310 Val Val Gly Leu Cys Gln Gly LeuThr Pro Val Asn Arg Gly Val 2315 2320 2325 Val Val Arg Thr Leu Ala GlnMet Gln Leu Asn Glu Glu Thr Ser 2330 2335 2340 Asn Ser Asn Gln Gly 2345

We claim:
 1. A nucleic acid encompassing a sequence selected from (a)the sequence as shown in SEQ ID NO: 1, (b) the sequence as shown inAccession Number AAF59156, (c) part-sequences of the sequences definedunder (a) and (b) which are at least 14 base pairs in length, (d)sequences which originate from insects or are derived therefrom andwhich hybridize with the sequences defined under (a) and (b) at ahybridization temperature of from 37° C. to 50° C., (e) sequences whichoriginate from insects or are derived therefrom and which have at least60% identity with the sequences defined under (a) and (b), (f) sequenceswhich are complementary to the sequence defined under (a) to (e), and(g) sequences which, owing to the degeneracy of the genetic code, encodethe same amino acid sequence as the sequences defined under (a) to (e).2. A vector encompassing at least one nucleic acid as claimed inclaim
 1. 3. A vector as claimed in claim 2, characterized in that thenucleic acid molecule is linked operably to regulatory sequences whichensure the expression of the nucleic acid in pro- or eukaryotic cells.4. A host cell encompassing a nucleic acid as claimed in claim 1 or avector as claimed in claim 2 or
 3. 5. A host cell as claimed in claim 4,characterized in that it is a pro- or eukaryotic cell.
 6. A host cell asclaimed in claim 5, characterized in that the prokaryotic cell isE.coli.
 7. A host cell as claimed in claim 5, characterized in that theeukaryotic cell is a mammalian cell or an insect cell.
 8. A polypeptidewith a sequence selected from a) the sequence with the biologicalactivity of an acetyl-CoA carboxylase isolated from Myzus persicae, b)the sequence as shown in SEQ ID NO: 2, c) the sequence encoded by anucleic acid of Accession Number AAF59156, d) part-sequences of thesequences mentioned under a) to c) which retain the biological activityof an acetyl-CoA carboxylase, e) sequences which originate from insectsor are derived therefrom and which have at least 60% identity with thesequences mentioned under a) to d).
 9. A method for preparing apolypeptide as claimed in claim 8, encompassing (a) culturing a hostcell as claimed in any of claims 4 to 7 under conditions which ensurethe expression of a nucleic acid as claimed in claim 1, and (b)obtaining the polypeptide from the cell or culture medium.
 10. Anantibody which reacts specifically with a polypeptide as claimed inclaim
 8. 11. The use of an insect polypeptide with the biologicalactivity of an acetyl-CoA carboxylase for identifying insecticidallyand/or acaricidally active compounds.
 12. The use as claimed in claim11, characterized in that the polypeptides are polypeptides as claimedin claim
 8. 13. The use of nucleic acids which encode insectpolypeptides with the biological activity of an acetyl-CoA carboxylasein methods for identifying modulators of these polypeptides.
 14. The useof nucleic acids which encode insect polypeptides with the biologicalactivity of an acetyl-CoA carboxylase for identifying substances whichmodify the expression of the polypeptides encoded by them.
 15. The useas claimed in claim 13 or 14, characterized in that fragments of genomicDNA or cDNA are involved.
 16. A method of finding a chemical compoundwhich binds to an insect polypeptide with the biological activity of anacetyl-CoA carboxylase, encompassing the following steps: (a) bringingan insect polypeptide with the biological activity of an acetyl-CoAcarboxylase or a host cell comprising such a polypeptide into contactwith a chemical compound or a mixture of chemical compounds underconditions which permit the interaction of one of these chemicalcompounds with the polypeptide, and (b) identifying the chemicalcompound which specifically binds to the polypeptide.
 17. A method ofidentifying substances which modulate the activity of acetyl-CoAcarboxylase from insects and/or Acarina, characterized in that a) thetest substance is brought into contact with acetyl-CoA carboxylase underconditions which permit an interaction of the test substance with theacetyl-CoA carboxylase, b) the interaction of the test substance whichhas taken place is detected by determining the ability of the acetyl-CoAcarboxylase to catalyze the biotin-dependent carboxylation ofacetyl-CoA, and c) the ability of the acetyl-CoA carboxylase to catalyzethe biotin-dependent carboxylation of acetyl-CoA in the presence of thetest substance is compared with its ability to catalyze thebiotin-dependent carboxylation of acetyl-CoA in the absence of a testsubstance.
 18. A method of finding a compound which modifies theexpression of insect polypeptides with the biological activity of anacetyl-CoA carboxylase, encompassing the following steps: (a) bringing ahost cell comprising a nucleic acid encoding an insect polypeptide withthe biological activity of an acetyl-CoA carboxylase into contact with achemical compound or a mixture of chemical compounds, (b) determiningthe polypeptide concentration, and (c) identifying the compound whichspecifically influences the expression of the polypeptide.
 19. The useof compounds of the formula (I)

in which Ar represents substituted aryl or hetaryl having at least oneortho-substituent, R represents H or acyl radicals, and A together withthe linked C atoms forms an optionally substituted 5- or 6-memberedcarbo- or heterocycle, suitable heteroatoms being, for example, N, Oand/or S, as inhibitors of insect acetyl-CoA carboxylase.
 21. The use ofcompounds of the formula (I) as claimed in claim 20 in methods asclaimed in claims 17 and
 18. 22. A modulator of acetyl-CoA carboxylasefrom insects and/or Acarina found by means of a method as claimed inclaim 16 or
 17. 23. An insecticidally and/or acaricidally activesubstance found by means of a method as claimed in claim 16 or 17.